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.

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.

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.

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.

[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.

Intracellular and extracellular redox environments surrounding redox-sensitive contrast agents under oxidative Atmosphere.

In vivo redox reactions of nitroxyl contrast agents in bile and blood under an oxidative atmosphere were investigated using normal healthy Wistar rats. Differences in intracellular and extracellular volumes in redox environments are discussed. Pharmacokinetic profiles of two nitroxyl contrast agents, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (carbamoyl-PROXYL), 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), in bile and blood were monitored by an electron paramagnetic resonance spectrometer when the rat was breathing 100% O(2) or was subcutaneously administrated 0.2 mmol/kg body weight of ferric citrate. Re-oxidation of hydroxylamines to nitroxyl radicals was caused in bile under 100% O(2) breathing, but not in blood. The administration of ferric citrate caused marked re-oxidation in bile, but a slight reduction in blood. Tissue H(2)O(2) level may partly play a role in the intracellular re-oxidation process. Tissue Fe(3+) concentration can work more effectively for the intracellular re-oxidation of hydroxylamines. The intracellular environment is susceptible to oxidation compared with the extracellular environment under conditions such as 100% O(2) breathing or iron overload.

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.