Effects of hydrogen-rich water and ascorbic acid treatment on spontaneously hypertensive rats.

Hydrogen-rich water (HW) has been suggested to possess antioxidant properties of value in treatments of lifestyle diseases and for prevention of latent pathologies. To date, the potential benefits of HW against the deleterious effects of excessive salt intake and hypertension have not been investigated. Here, we first examined the effects of HW or HW supplemented with 0.1% ascorbic acid (HWA) on spontaneously hypertensive rats (SHR) that had been fed a normal diet. In comparison to control rats given distilled water (DW), we found that HW did not significantly influence systolic blood pressure (SBP) or diastolic blood pressure (DBP) in SHR; however, the increase in SBP and DBP were inhibited in the HWA group. Next, four groups of SHR were given DW, 0.1% ascorbic acid-added DW (DWA), HW, or HWA in combination with a 4% NaCl-added diet. SHR fed the 4% NaCl-added diet showed increased hypertension; HWA treatment resulted in a significant reduction in blood pressure. The HWA group tended to have lower plasma angiotensin II levels than the DW group. In addition, urinary volumes and urinary sodium levels were significantly lower in the HWA group than the DW group. Urinary isoprostane, an oxidative stress marker, was also significantly lower in the HWA group, suggesting that the inhibitory effect of HWA on blood pressure elevation was caused by a reduction in oxidative stress. These findings suggest a synergistic interaction between HW and ascorbic acid, and also suggest that HWA ingestion has potential for prevention of hypertension.

Analysis of the antioxidative function of the radioprotective Japanese traditional (Kampo) medicine, hangeshashinto, in an aqueous phase.

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH)-scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O(2) (•-))-scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O(2) (•-). Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.

Irx3 and Pax6 establish differential competence for Shh-mediated induction of GABAergic and glutamatergic neurons of the thalamus.

During embryonic development, the presumptive GABAergic rostral thalamus (rTh) and glutamatergic caudal thalamus (cTh) are induced by Sonic hedgehog (Shh) signaling from the zona limitans intrathalamica (ZLI) at the rostral border of the thalamic primordium. We found that these inductions are limited to the neuroepithelium between the ZLI and the forebrain-midbrain boundary, suggesting a prepattern that limits thalamic competence. We hypothesized that this prepattern is established by the overlapping expression of two transcription factors: Iroquois-related homeobox gene 3 (Irx3) posterior to the ZLI, and paired box gene 6 (Pax6) anterior to the forebrain-midbrain boundary. Consistent with this assumption, we show that misexpression of Irx3 in the prethalamus or telencephalon results in ectopic induction of thalamic markers in response to Shh, that it functions as a transcriptional repressor in this context, and that antagonizing its function in the diencephalon attenuates thalamic specification. Similarly, misexpression of Pax6 in the midbrain together with Shh pathway activation results in ectopic induction of cTh markers in clusters of cells that fail to integrate into tectal layers and of atypical long-range projections, whereas antagonizing Pax6 function in the thalamus disrupts cTh formation. However, rTh markers are negatively regulated by Pax6, which itself is down-regulated by Shh from the ZLI in this area. Our results demonstrate that the combinatorial expression of Irx3 and Pax6 endows cells with the competence for cTh formation, whereas Shh-mediated down-regulation of Pax6 is required for rTh formation. Thus, thalamus induction and patterning depends both on a prepattern of Irx3 and Pax6 expression that establishes differential cellular competence and on Shh signaling from the ZLI organizer.

Biliary excretion of essential trace elements in rats under oxidative stress caused by selenium deficiency.

The excretion of essential trace elements, namely, Se, Sr, As, Mn, Co, V, Fe, and Zn into the bile of Se-deficient (SeD) Wistar male rats was studied using the multitracer (MT) technique, and instrumental neutron activation analysis (INAA). Normal and Se-control (SeC) rat groups were used as reference groups to compare the effects of Se levels on the behaviors of the essential trace elements. The excretion (% dose) of Se, Sr, As, Mn, Co, and V increased with Se levels in the liver. The biliary excretion of Mn and As dramatically enhanced for SeC rats compared with SeD rats, while that of V accelerated a little for SeC rats. The radioactivity levels of (59)Fe and (65)Zn in the MT tracer solution were insufficient to measure their excretion into bile. The role of glutathione and bilirubin for biliary excretion of the metals was discussed in relation to Se levels in rat liver.

Y-box binding protein 1 is up-regulated in proliferative breast cancer and its inhibition deregulates the cell cycle.

The Y-box-binding protein 1 (YB-1), a member of the cold-shock domain RNA-and DNA-binding protein family, has pleiotropic functions such as regulation of the cell cycle. The aim of this study was to evaluate if YB-1 is a proliferative marker in breast cancer and elucidate potential downstream targets involved in YB-1-mediated cell cycle regulation using RNA interference technology. YB-1 protein expression was evaluated in tissue microarrays of 131 breast invasive ductal carcinomas by immunohistochemistry, while the YB-1 gene expression profile was evaluated in the T-47D, MDA-MB-231, ZR-75-1 and MCF7 breast cancer cell lines. Silencing of the YB-1 gene in T-47D breast cancer cells was performed using siRNA and the effects of down-regulation of YB-1 on cell growth and regulation of the cell cycle were ascertained. A focused panel of 84 genes involved in cell cycle progression was also examined. In tissue microarrays, YB-1 expression was shown to be associated with proliferating cell nuclear antigen (PCNA) immunostaining. siRNA-mediated silencing of the YB-1 gene inhibited cell proliferation and induced G1 phase cell cycle arrest in T-47D breast cancer cells. Knockdown of the YB-1 gene induced up-regulation of two genes which contribute to G1-arrest (RAD9A and CDKN3 genes) and down-regulation of ten genes associated with positive regulation of the cell cycle (SKP2, SUMO1, ANAPC4, CCNB1, CKS2, MNAT1, CDC20, RBBP8, KPNA2 and CCNC genes). The data obtained from the tissue microarrays and cell lines provide evidence that YB-1 is a reliable marker of cell proliferation and possibly a potential molecular target in breast cancer therapy.

An EPR method for estimating activity of antioxidants in mouse skin using an anthralin-derived radical model.

Inhibitory effects of intravenously or orally administered antioxidants on the anthralin-derived radical generated in skin (mainly in the epidermis) of living mice by ultraviolet-A (UVA) irradiation were estimated. Anthralin was applied to the dorsal skin of living mice and the mice were then exposed to UVA. The EPR signal intensity in skin tissue strips obtained from mice after anthralin-UVA treatment was measured by an X-band EPR spectrometer. Several common antioxidants such as ascorbate, glutathione and Trolox (a vitamin E analogue) intravenously administered to mice reduced anthralin-derived radical generation. Trolox showed the most prolonged and powerful effect. Intravenous injection of a clinically used cerebral neuroprotective drug, Edarabone (Radicut), also showed depletion for the anthralin-derived radical. Oral administration of a commercialized nutritional supplement (a cocktail of 17 herbals and vitamins) also attenuated the anthralin-derived radical. The anthralin-UVA treatment model for antioxidant activity in the epidermis is a potentially feasible method to estimate activity of antioxidants in the body.

The effect of long-running severe selenium-deficiency on the amount of iron and zinc in the organs of rats.

The amounts of selenium (Se), iron (Fe), and zinc (Zn) in the liver, kidney, and spleen as a function of age of rats measured using instrumental neutron activation analysis were compared between Se-deficient (SeD) rats and normal rats. The SeD model rats can live for more than 50 weeks. The effect of Se-deficinecy in rats might be weak, compared to the marked malfunction of GSH-Px. The SeD rats can be considered as a model of nonlethal chronic oxidative stress. Fluctuations of Fe and Zn in the liver of Se-deficient rats were observed. The amount of redox-relating minerals, such as Fe and Zn, in SeD rat organs is changeable depending on the age.

Nitroxyl radicals as low toxic spin-labels for non-invasive magnetic resonance imaging of blood-brain barrier permeability for conventional therapeutics.

The present study describes a novel non-radioactive methodology for in vivo non-invasive, real-time imaging of blood-brain barrier (BBB) permeability for conventional drugs, using nitroxyl radicals as spin-labels and magnetic resonance imaging (MRI).

MR assessment of changes of tumor in response to hyperbaric oxygen treatment.

Enhancement of image intensity, using the T1-weighted spoiled gradient-echo (SPGR) sequence, was measured in SCC tumor implanted in the flank of C3H mice while they were subjected to several types of oxygenation challenges inside a hyperbaric chamber designed and constructed to fit in an MRI resonator. The central portions of the tumor gave a positive enhancement, while the periphery showed signal reduction during both normobaric (NBO) and hyperbaric (HBO) oxygen challenges. In the contralateral normal leg, nearly 70% of the region showed a decrease in intensity, and the rest showed a positive enhancement. The positive signal enhancement was markedly greater under HBO compared to NBO. Calculated R1, R2, and M0 maps from multivariate fitting of images acquired by a multislice multiecho (MSME) sequence with variable TR before, during, and after HBO treatment confirm that the source of SPGR signal enhancement in the tumor is associated with shortening of T1.

Importance of volume limitation for tissue redox status measurements using nitroxyl contrast agents: a comparison of X-band EPR bile flow monitoring (BFM) method and 300 MHz in vivo EPR measurement.

Methods proposed for in vivo redox status estimation, X-band (9.4 GHz) electron paramagnetic resonance (EPR) bile flow monitoring (BFM) and 300 MHz in vivo EPR measurement, were compared. The spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was utilized for both methods, due to its suitable lipophilicity. EPR signal decay of a nitroxyl spin probe in the bile flow and in the liver region (upper abdomen) of several rat groups with different selenium status were measured by both the BFM and the in vivo EPR method, respectively. The nitroxyl radical clearance measured with in vivo EPR method may be affected not only by the redox status in the liver but also by information from other tissues in the measured region of the rat. On the other hand, the time course of nitroxyl radical level in the bile flow of rats was found to be a reliable index of redox status. Measurement site and/or volume limitation, which was achieved by the BFM method in this paper, is quite important in estimating reasonable EPR signal decay information as an index of tissue/organ redox status.

Electron paramagnetic resonance decay constant and oxidative stresses in liver microsomes of the selenium-deficient rat.

The free radical-reducing activity and the membrane fluidity of liver microsomes from selenium-deficient (SeD) rats were examined by means of electron paramagnetic resonance (EPR) spin label method using nitroxyl-labeled stearic acids. Our findings show that the membrane fluidity and lipid peroxidation levels in SeD rat liver microsome were relatively unchanged compared with normal rat. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity. The nitroxyl spin probes are substrates for reduction-relating cytochrome P-450. Previous in vivo studies suggested that the total liver free radical reduction activity in SeD rat was decreased. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity, and the reduction rate of nitroxyl radical existing at shallow depth in membrane was increased. Selenium-deficient rats experienced an increase in hydrogen peroxide (H2O2) due to a pronounced loss of glutathione peroxidase (GSH-Px) activity. This masked the overall reduction rate of the nitroxyl spin probe by reoxidation of the hydroxylamine form. Although the SeD condition caused induction of liver cytochrome P-450 and chronic increased H2O2, this did not result in oxidative liver damage. An increased level of glutathione in SeD liver was also evident, likely due to the absence of GSH-Px activity. Using the EPR spin label method, we have shown that SeD causes complicated redox changes in the liver, notably, alterations in the levels of cytochrome P-450 and GSH-Px systems.

In vivo multitracer analysis technique: screening of radioactive probes for noninvasive measurement of physiological functions in experimental animals.

A novel screening experiment, to find radioactive probes for non-invasive measurements of physiological functions in experimental animals, was tested using the in vivo multitracer analysis technique. The details of the efficiency of the detector settings used in the in vivo multitracer analysis technique were examined by both computer simulations and practical measurements. Multiple radioactive isotopes, i.e. multitracer, were prepared by irradiating a silver foil target with a heavy ion beam at the RIKEN ring cyclotron. After chemical separation of the silver target, the multitracer was finally dissolved in isotonic citrate buffer. The multitracer solution was intravenously injected into rats. Using a gamma-ray detector equipped with a well-defined slit, the collimated gamma-rays from the upper abdomen of living rats were measured. After correction of detection efficiencies, it was possible to compare the distribution of radioactive elements between two groups of rats different in body weight. The in vivo measurement showed that the tissue substantial volume of the selenium-deficient (SeD) rat liver increased compared to normal rats. The possibility of a functional estimation of tissue/blood volume for living rats was proposed based on the characteristic in vivo distribution of 74As, 83Rb and 103Ru.

Correlation between ketone body level in selenium-deficient rats and oxidative damages.

The age dependence of ketone body levels (KBLs) and oxidative damages in selenium-deficient (SeD) and normal rats were compared. The feeding SeD diets gave ketogenesis and higher KBLs especially in younger rats. However, KBLs in SeD rats seemed to decrease with their age. Feeding 0.1 mg/kg Se in water with SeD diet did not affect the KBLs in young (8 week old) rats, whereas the addition of Se reduced the KBLs in older (20 week old) rats. Blood KBLs showed some correlations with tissue damage. TBARSs showed no correlations with the tissue damages and KBLs when the values were compared between the same age, while better correlation was obtained between urinary KBLs of 6-20 week old normal rats and the liver TBARSs of 4-16 week old normal rats. The oxidative injury might induce liver damage with some delay. SeD rat kidney TBARS levels normalized by protein had some correlations with BUN and blood KBL. Kidney may be sensitive to the oxidative stresses and/or injuries. Tissue damages of SeD rats decreased with age. In contrast, oxidative injuries might be gradually accumulated in normal rat tissue. Oxidative stress can be visible by gradual accumulation of small damages during the aging, while large stress in young rats can be buffered and masked. The aging based accumulation of oxidative injuries might also be correlated with KBLs, while it might not give notable tissue damages.

Effect of hydrogen peroxide in redox status estimation using nitroxyl spin probe.

A procedure for estimating in vivo redox status using EPR and a hydrogen peroxide (H(2)O(2))-dependent spin probe method is described. The mechanism of decreasing spin clearance in the selenium-deficient (SeD) rat is discussed. The in vivo decay constant of the nitroxyl spin probe in the liver region of SeD rats appeared to be slightly lower that of the selenium-adequate control (SeC) group, and was significantly smaller than that of normal rats. Bile H(2)O(2) levels in normal rats were significantly lower than those in SeD rats. The in vivo decay constant of the spin probe in SeD rats depended on the bile H(2)O(2) level. Furthermore, H(2)O(2) was detected in the bile in all SeD rats, whereas bile H(2)O(2) could be detected in only half of the normal rats. It was found that the in vivo decay constant of the spin probe in normal rats also depended on whether bile H(2)O(2) was detected or not. In vivo decay constants were smaller in rats subjected to the surgical operation than in the nonoperated groups. The EPR signal of the nitroxyl radical in the liver homogenate was increased by addition of H(2)O(2), which was administered 30 min before the rat was killed. It appears that H(2)O(2) can oxidize the hydroxylamine formed following reduction of the spin probe in the liver.

C-propeptide region of human pro alpha 1 type 1 collagen interacts with thioredoxin.

Thioredoxin (TRX) is one of major components of thiol reducing systems. To investigate the molecular mechanism of TRX function in the lung tissue, we screened a human lung epithelial cell cDNA library for TRX-binding protein by yeast two-hybrid systems. We isolated a plasmid containing C-propeptide region of human pro alpha 1 type 1 collagen (CP-pro alpha 1(1)). CP-pro alpha 1(1) stably binds to wild type TRX but not to mutant TRX, in which redox-active cysteine residues are substituted. Failure of the interaction of mutant TRX with CP-pro alpha 1(1) was confirmed in yeast two-hybrid systems. The CP-pro alpha 1(1)/TRX interaction was increased by dithiothreitol treatment, but was markedly inhibited by hydrogen peroxide or diamide treatment. These data showed that the reducing status of TRX active site cysteine residues is important for the TRX-CP-pro alpha 1(1) interaction, indicating that collagen biosynthesis is under the regulation of TRX-dependent redox control.

[Relation of bile hydrogen peroxide level and liver super oxide dismutase activity in selenium-deficient rats].

The relationship of hydrogen peroxide (H2O2) levels in bile with liver SOD and GSH-Px activity in selenium (Se)-deficient rats is discussed. Normal rats and 7 groups of rats fed a Se-deficient diet with different feeding periods were examined. H2O2 levels in bile were measured using the spin-trapping method with electron spin resonance (ESR). Bile H2O2 levels in the initial stage (20-60 min from start of the cannulation) of measurement were increased depending on the length of the feeding period with the Se-deficient diet and absence of Se. Bile H2O2 levels in the later stage (60-120 min) of measurement first increased with the length of feeding with the Se-deficient diet and then decreased with longer feeding periods. Bile H2O2 levels immediately after the operation were relatively low in almost all cases. The operation may result in oxidative stress to generate H2O2. Liver GSH-Px activity decreased depending on the length of the feeding period with the Se-deficient diet and existence of Se. Liver SOD activity increased in Se-deficient groups. It is suggested that the H2O2 levels in bile are related to decreased GSH-Px activity, SOD activity, and also the oxidative stress caused by surgery. Therefore the H2O2 levels in bile can be used as an index of sensitivity to oxidative stress. Although severe oxidative stress may decrease SOD activity, Se deficiency can induce liver SOD activity.

[Noninvasive detection of dynamics of various elements in the upper abdomen of selenium-deficient rat using multitracer analysis technique].

The advantages of a technique as a diagnostic tool for examining the distribution of bio-trace elements in the living body are discussed. Time courses of the distribution of Be, V, Mn, Fe, Co, Zn, As, Se, Rb, Sr, and Y in the upper abdomen of living selenium-deficient rats were examined using the in vivo multitracer analysis technique. The dynamics of the elements were estimated by comparison with the distribution of As. Almost all As was taken up by red blood cells. The present findings of a decrease in Se and increase in Co in the liver of Se-deficient rats are in good agreement with the previous data that showed a decrease in Se and increase in Co uptake into the liver cell fraction of Se-deficient rats. Although the normalized uptake rate and the relative distribution of Co 48 h after administration increased in Se-deficient rats, the early level of the relative distribution of Co was not lower compared with that in the normal group. This suggests that the high level of the normalized uptake rate and the relative distribution of Co in Se-deficient rats were affected by the decreasing excretion rate rather than by the increasing uptake rate of Co. The plateau level of relative distribution of Se in the Se-deficient rats is lower than that in normal rats, suggesting that the lower levels of the normalized uptake rate and relative distribution of Se in Se-deficient rats were due to the decreased uptake rate of the element.

[Measurement of contents in organs of selenium- or vitamin E-deficient rat using instrumental neutron activation analysis].

The contents of iron (Fe), cobalt (Co), zinc (Zn), and selenium (Se) in the organs (liver, kidney, spleen, heart, lung, and brain) and the liver cell fractions (nuclear, mitochondrial, microsomal, and cytosolic fractions) of Se- or vitamin E (VE)-deficient rats were measured using instrumental neutron activation analysis (INAA). The contents of Fe in the liver of Se-deficient rats, and in the liver and the spleen of VE-deficient rats were increased compared with those in normal rats. Fe contents increased mainly in the microsomal fraction. Contents of Co in the organs and liver cell fractions of Se- and VE-deficient rats were markedly low, reflecting the Co contents in both diets. Contents of Zn in the organs and liver cell fractions of Se- and VE-deficient rats decreased to 60-80% of the contents in normal rats. The Se contents in Se-deficient rat organs except for the kidney, spleen, and brain were below the detectable level under the present conditions. Se contents in VE-deficient rat decreased to 50-80% of those in normal rats in all organs and fractions. It is suggested that oxidative stress due to Se- or VE-deficiency affects the dynamics of Fe and Zn.

Evaluation of oxidative damage in the liver of selenium-deficient rats.

Previous studies have implied a relationship between Se-deficiency and oxidative stress. In the present study, the occurrence of oxidative stress due to Se-deficiency was investigated by evaluating the age dependence of growth and indices of oxidative damage for the liver of Se-deficient (SeD) rats. The ratios of liver weight to body weight of the SeD rats were greater than those of the normal rats. The values of AST and ALT (clinical indices of liver damage) were higher in the SeD rats than the normal ones especially in the young (6-12 weeks of age). The TBARS level of the 4-week-old SeD group were higher than the normal group while the level decreased with age. Conversely, the TBARS level of the normal group gradually increased and became higher than SeD group in older rats (12-20 weeks of age). Vitamin E rather than vitamin C may be consumed during oxidative stress due to Se-deficiency. Damage induced by Se-deficiency may be related to growth and the mechanisms of this damage may alter with age.