Journal of Clinical Biochemistry and Nutrition Vol. 56(2015) No. 1

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O₂^•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q₁₀) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

Treatments with nonsteroidal anti-inflammatory drugs (NSAIDs) have increased the number of patients with gastrointestinal complications. Qing Dai has been traditionally used in Chinese herbal medicine for various inflammatory diseases such as ulcerative colitis. We previously reported that Qing Dai suppressed inflammations by scavenging reactive oxygen species (ROS) in ulcerative colitis patients. Thus, Qing Dai can attenuate the production of ROS, which play an important role in NSAID-induced gastrointestinal injuries. In this study, we aimed to elucidate whether Qing Dai decreased mitochondrial ROS production in NSAID-treated gastrointestinal cells by examining cellular injury, mitochondrial membrane potentials, and ROS production with specific fluorescent indicators. We also performed electron paramagnetic resonance measurement in isolated mitochondria with a spin-trapping reagent (CYPMPO or DMPO). Treatments with indomethacin and aspirin induced cellular injury and mitochondrial impairment in the gastrointestinal cells. Under these conditions, mitochondrial alterations were observed on electron microscopy. Qing Dai prevented these complications by suppressing ROS production in gastrointestinal cells. These results indicate that Qing Dai attenuated the ROS production from the NSAID-induced mitochondrial alteration in the gastrointestinal epithelial cells. Qing Dai treatment may be considered effective for the prevention NSAID-induced gastrointestinal injury.

The twin character of reactive oxygen species is substantiated by a growing body of evidence that reactive oxygen species within cells act as inducers and accelerators of the oncogenic phenotype of cancer cells, while reactive oxygen species can also induce cancer cell death and can therefore function as anti-tumorigenic species. The aim of this study was to assess a possible influence of xanthine/xanthine oxidase on the proliferation of colorectal cancer cell line Caco-2. xanthine/xanthine oxidase (2.5 µM/0.25 mU/ml–25 µM/2.5 mU/ml) dose-dependently inhibited the proliferation of Caco-2 cells. Experiments utilizing reactive oxygen species scavengers (superoxide dismutase, catalase and mannitol) and exogenous hydrogen peroxide revealed a major role of hydrogen peroxide in the xanthine/xanthine oxidase effect. Investigations utilizing annexin V-fluorescein/PI assay using flow cytometry, and the lactate dehydrogenase extracellular release assay indicated that hydrogen peroxide induced necrosis, but not apoptosis, in Caco-2 cells. These results suggest that hydrogen peroxide generated by xanthine/xanthine oxidase has the potential to suppress colorectal cancer cell proliferation.

The effect of rebamipide, a mucosal protective drug, on small intestinal mucosal injury caused by indomethacin was examined using a rat model. Indomethacin administration (10 mg/kg, p.o.) induced intestinal mucosal injury was accompanied by an increase in the numbers of intestinal bacteria particularly Enterobacteriaceae in the jejunum and ileum. Rebamipide (30 and 100 mg/kg, p.o., given 5 times) was shown to inhibit the indomethacin-induced small intestinal mucosal injury and decreased the number of Enterococcaceae and Enterobacteriaceae in the jejunal mucosa to normal levels. It was also shown that the detection rate of segmented filamentous bacteria was increased by rebamipide. PCR array analysis of genes related to inflammation, oxidative stress and wound healing showed that indomethacin induced upregulation and downregulation of 14 and 3 genes, respectively in the rat jejunal mucosa by more than 5-fold compared to that of normal rats. Rebamipide suppressed the upregulated gene expression of TNFα and Duox2 in a dose-dependent manner. In conclusion, our study confirmed that disturbance of intestinal microbiota plays a crucial role in indomethacin-induced small intestinal mucosal injury, and suggests that rebamipide could be used as prophylaxis against non-steroidal anti-inflammatory drugs -induced gastrointestinal mucosal injury, by modulating microbiota and suppressing mucosal inflammation in the small intestine.

This study investigated the effect and mechanism of pre-germinated brown rice (PGBR) prevented hyperglycemia in C57BL/6J mice fed high-fat-diet (HFD). Normal six-week-old mice were randomly divided into three groups. Group 1 was fed standard-regular-diet (SRD) and group 2 was fed HFD for 16 weeks. In group 3, the mice were fed a HFD with its carbohydrate replaced with PGBR for 16 weeks. Comparing the SRD and HFD groups, we found the HFD group had higher blood pressure, higher concentrations of blood glucose and HbA1c. The HFD group had less protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), glucose transporter-4 (GLUT-4) and glucokinase (GCK) and greater expression of glucogen synthase kinase (GSK) in skeletal muscle. The HFD group also had less expression of IR, serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB), AMP-activated protein kinase (AMPK), GCK and peroxisome proliferator-activated receptor γ (PPARγ) in liver. In the HFD + PGBR group, the PGBR could reverse the disorders of blood pressure, blood glucose, HbA1c and increase insulin concentration. PGBR increased the IR, IRS-1, PI3K, Akt, GLUT-1 and GLUT-4 proteins, and ameliorated AMPK, GCK, GSK and PPARγ proteins. Together, PGBR prevented HFD-induced hyperglycemia through improving insulin levels, insulin receptor, glucose transporters and enhancing glucose metabolism.

High serum phosphorus (P) impairs endothelial function by increasing oxidative stress and decreasing nitric oxide production. Serum P levels fluctuate due to circadian rhythms or dietary P intake in healthy people and due to dialysis in end-stage chronic kidney disease patients. Here we examined whether fluctuating plasma P caused by changes in dietary P intake may be involved in endothelial dysfunction, resulting in increased cardiovascular risk. Rats were fed a diet containing 0.6% P for 16 days (control group), or a diet alternating between 0.02% P and 1.2% P (LH group) or between 1.2% P and 0.02% P (HL group) every 2 days; the total amount of P intake among the groups during the feeding period was similar. In the LH and HL groups, endothelial-dependent vasodilation significantly decreased plasma 8-(OH)dG level significantly increased, and the expression of inflammatory factors such as MCP-1 increased in the endothelium as compared with the control group. These data indicate that repetitive fluctuations of plasma P caused by varying dietary P intake can impair endothelial function via increased oxidative stress and inflammatory response. Taken together, these results suggest that habitual fluctuation of dietary P intake might be a cause of cardiovascular disease through endothelial dysfunction, especially in chronic kidney disease patients.

Peyer’s patches are nodules that play a central role in intestinal immunity. Few studies demonstrate the relationship between the number of Peyer’s patches and intestinal polyps. Here we identify a statistically significant inverse correlation between the quantity of Peyer’s patches and of the development of intestinal polyps in Apc^Min/+ mice, which are a useful model to clarify the role of Peyer’s patches in intestinal tumorigenesis. Using this model, we increased the number of Peyer’s patches using 0.1% and 1% corn husk arabinoxylan through feed. Intestinal polyp formation significantly decreased, concomitant with an increase in Peyer’s patches development (n = 12/group). In Aly^−/−Apc^Min/+ mice (negative control; no Peyer’s patches) there was no change in the amount of intestinal polyps (n = 10/group). Immune reaction following corn husk arabinoxylan treatment was measured by cytokine array. Increasing the number of Peyer’s patches decreased interleukin-17 production, which showed a dose dependent correlation with transcription factor/lymphoid enhancer-binding factor. This study identified a relationship between levels of Peyer’s patches and intestinal polyp formation, partly explained by the involvement of interleukin-17 production and β-catenin signaling in Apc^Min/+ mice.

DNA damages and antioxidant status was assessed after 8 weeks of purple grape juice supplementation in male smokers depending on the glutathione S-transferase polymorphisms. Ninety-five smokers consumed 480 ml of purple grape juice for 8 weeks. The blood samples were collected before and after supplementation to measure lymphocyte DNA damages, plasma antioxidants, conjugated diene, and the erythrocyte antioxidant enzymes. The diastolic pressure, lymphocyte DNA damage, and plasma conjugated diene were significantly decreased but the plasma γ-tocopherol was increased in GSTM1-null genotype, while increased blood glutathione and decreased lymphocyte DNA damage were observed in GSTM1-present genotype. In case of GSTT1 on the other hand, the decrease in diastolic pressure and lymphocyte DNA damage was observed in both null types and present types, but the erythrocyte catalase activity was decreased in GSTT1-null type and the plasma vitamin C level was increased in GSTT1-present type, suggesting that, the antioxidant effect of grape juice was greater in GSTT1-present type compared to GSTT1-null type. The intakes of 8-week purple grape juice affected diastolic blood pressures, DNA damage reductions and antioxidant status in smokers, mainly greater in GSTM1-null type and GSTT1-present type.

Amniotic fluid contains numerous biomolecules derived from fetus and mother, thus providing precious information on pregnancy. Here, we evaluated oxidative stress of human amniotic fluid and measured the concentration of catalytic Fe(II). Amniotic fluid samples were collected with consent from a total of 89 subjects in Nagoya University Hospital, under necessary medical interventions: normal pregnancy at term, normal pregnancy at the 2nd trimester, preterm delivery with maternal disorders but without fetal disorders, congenital diaphragmatic hernia, fetal growth restriction, pregnancy-induced hypertension, gestational diabetes mellitus, Down syndrome and trisomy 18. Catalytic Fe(II) and oxidative stress markers (8-hydroxy-2'-deoxyguanosine, 8-OHdG; dityrosine) were determined with RhoNox-1 and specific antibodies, respectively, using plate assays. Levels of 8-OHdG and dityrosine were higher in the 3rd trimester compared with the 2nd trimester in normal subjects, and the abnormal groups generally showed lower levels than the controls, thus suggesting that they represent fetal metabolic activities. In contrast, catalytic Fe(II) was higher in the 2nd trimester than the 3rd trimester in the normal subjects, and overall the abnormal groups showed higher levels than the controls, suggesting that high catalytic Fe(II) at late gestation reflects fetal pathologic alterations. Notably, products of H₂O₂ and catalytic Fe(II) remained almost constant in amniotic fluid.

The objective of this study is to valuate two biomarkers that may guide nutritional assessment during follow up after intestinal transplantation. We performed a retrospective study on prospectively collected data of insulin-like growth factor-1 (IGF-1) and effluent calprotectin in patients undergoing intestinal transplantation. Optimal nutritional status (ONS) was defined by using the Malnutrition Universal Screening Tool (MUST). IGF-1 and calprotectin were correlated with ONS by Pearson correlation. Eighteen cadaveric intestinal transplants were performed over 1,650 days (median follow up 425 days, range 29–1,650 days). Mean IGF-1 and calprotectin were significantly associated with independent nutrition. Seven patients became malnourished on one or more occasions. During malnutrition the mean IGF-1 was 22 ± 14 ng/ml and calprotectin 1,597 ± 1,055 mcg/g. Mean weight during episodes of malnutrition changed from 64.77 ± 8.76 kg to 59.05 ± 8.5 kg (–8.9 ± 1.25%). Both IGF-1 and calprotectin negatively correlated with ONS (Pearson’s r, –0.612, p = 0.014). Patients broadly aligned with three groups: nutritionally replete (normal IGF-1 and normal calprotectin), nutritionally equivocal (normal or low normal IGF-1 and high calprotectin), and malnourished (low IGF-1 and high calprotectin). Patients with low IGF-1 and high calprotectin may have a benign clinical presentation. However it is in their interests to have parenteral nutrition restarted pending further investigation.

Hyaluronan (HA) has been increasingly used as a dietary supplement to improve the skin. However, the effect of ingested HA may depend on its molecular weight (MW) because its physiological activities in the body vary with its MW. In this study, we examined the effects of ingested HA with varying MW on the skin. In this randomized, double blind, placebo controlled study, 61 subjects with dry skin received oral HA (120 mg/day), of MWs 800 k and 300 k or placebo, for 6 weeks. The skin moisture contents of the first two groups increased more than those of the placebo group during the ingestion period. In addition, group HA 300 k exhibited significant improvements in skin moisture content 2 weeks after ingestion ended compared with the placebo group. A questionnaire survey about subjective facial aging symptoms showed that the HA treated groups exhibited significantly improved the skin condition compared with the placebo treated group. Furthermore, dermatologists objectively evaluated the clinical symptoms of the facial and whole body skin, showing that no adverse events were related to daily ingestion of HA. This study shows that both of ingesting HAs (MWs 800 k and 300 k) improved the skin condition by increasing the moisture content.

The cellular mechanisms involved in the development of silicosis have not been fully elucidated. This study aimed to examine influence of silica-induced lung injury on autophagy. Suspensions of crystalline silica particles were administered transnasally to C57BL/6j mice. Immunohistochemical examination for Fas and p62 protein expression was performed using lung tissue specimens. Two-dimensional and quantitative analysis of silica deposits in the lungs were performed in situ using lung tissue sections by an in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis system, which was based on irrradiation of specimens with a proton ion microbeam. Quantitative analysis showed a significant increase of iron levels on silica particles (assessed as the ratio of Fe relative to Si) on day 56 compared with day 7 (p<0.05). Fas and p62 were expressed by histiocytes in granulomas on day 7, and the expressions persisted for day 56. Fas- and p62-expressing histiocytes were co-localized in granulomas with silica particles that showed an increase of iron levels on silica particles in mouse lungs. Iron complexed with silica induces apoptosis, and may lead to dysregulations of autophagy in histiocytes of granulomas, and these mechanisms may contribute to granuloma development and progression in silicosis.