Mieko Hayakawa

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress.

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ekd) for AIPH was 2.6 × 10-6/s at 37°C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2α (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.

Characterization of Cellular Uptake and Distribution of Vitamin E

Abstract: We previously reported that tocotrienols acted as more potent inhibitors against selenium deficiency‐induced cell death than the corresponding tocopherol isoforms (J. Biol. Chem. 2003;278:39428‐39434). In the present study, we first compared the differences in the cellular uptake between α‐tocopherol (α‐Toc) and α‐tocotrienol (α‐Toc‐3). The initial rate of cellular uptake of α‐Toc‐3 was 70‐fold higher than that of α‐Toc. Subcellular fractionation analysis of α‐Toc‐3 and α‐Toc‐fortified cells showed similar cellular distribution of these antioxidants, which was directly proportional to the lipid distribution. The cells containing similar amounts of α‐Toc‐3 and α‐Toc showed similar resistance against the oxidative stress caused by peroxides. These results suggest that the apparent higher cytoprotective effect of α‐Toc‐3 than α‐Toc is primarily ascribed to its higher cellular uptake.

Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells

Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested.

Distribution of tocopherols and tocotrienols to rat ocular tissues after topical ophthalmic administration

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. Chemically, tocopherols and tocotrienols are closely related; however, it has been observed that they have widely varying degrees of biological effectiveness. The present study has been carried out in an attempt to deepen our understanding of whether there is a significant difference in distribution between tocopherol and tocotrienol homologs to rat eye tissues. Rats were administered 5 μl of pure tocopherol or tocotrienol to each eye once a day for 4 d. Various tissues of the eyes were separated and analyzed for tocopherol and tocotrienol concentrations. The concentration of α-tocotrienol increased markedly in every tissue to which it was administered; however, no significant increase was observed in the case of α-tocopherol. The intraocular penetration of γ-tocopherol and γ-tocotrienol did not differ significantly. Additionally, a significant increase in total vitamin E concentration was observed in ocular tissues, including crystalline lens, neural retina, and eye cup, with topical administration using a relatively small amount (5 μL) of vitamin E, whereas no significant increase was observed when the same amount of vitamin E was administered orally. Topical administration of tocotrienols is thus an effective way to increase ocular tissue vitamin E concentration.

The role of α-tocopherol in motor hypofunction with aging in α-tocopherol transfer protein knockout mice as assessed by oxidative stress biomarkers.

It has been hypothesized that oxidative stress plays a key role in aging. In order to elucidate the role of the antioxidant network - including alpha-tocopherol (alphaT) and alphaT transfer protein - in aging in vivo, alpha-tocopherol transfer protein knockout (alphaTTP(-/-)) mice were fed a vitamin-E-depleted diet, and wild-type (WT) mice were fed a diet containing 0.002 wt.% alphaT from the age of 3 months to 1 1/2 years. The lipid oxidation markers total hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F(2)alpha, and antioxidant levels in the blood, liver and brain were measured at 3, 6, 12 and 18 months. tHODE levels in the plasma of alphaTTP(-/-) mice were elevated at 6 months compared to 3 months, and were significantly higher those in WT mice, although they decreased thereafter. On the other hand, tHODE levels in the liver and brain were constantly higher in alphaTTP(-/-) mice than in WT mice. Motor activities decreased with aging in both mouse types; however, those in the alphaTTP(-/-) mice were lower than those in the WT mice. It is intriguing to note that motor activities were significantly correlated with the stereoisomer ratio (Z,E/E,E) of HODE, which is a measure of antioxidant capacity in vivo, in the plasma, in the liver and even in the brain, but not with other factors such as antioxidant levels. In summary, using the biomarker tHODE and its stereoisomer ratio, we demonstrated that alphaT depletion was associated with a decrease in motor function, and that this may be primarily attributable to a decrease in the total antioxidant capacity in vivo.

A Gene Expression Profiling Approach to Study the Influence of Ultrafine Particles on Rat Lungs

In recent years, industrial or commercial products incorporating nanomaterials have triggered concerns for human health. Manufactured nanomaterials are unstable and tend to form secondary particles by agglomeration. Little is known about the cytotoxicity induced by nano-sized secondary particles dispersed in aqueous solution. In the present study, we have attempted to disperse ultrafine nickel oxide particles in water (Uf-NiO), characterize the physicochemical properties, and instill into the trachea of rat lungs. Analysis by inductively couple plasma mass spectrometry (ICP-MS) and transmission electron microscope (TEM) revealed that the Uf-NiO particles began to be cleared from the lungs immediately after treatment, and that low levels of the particles were present at 6 months post-instillation. Genome-wide expression analysis using DNA microarray revealed that intratracheal instillation of Uf-NiO particles led to a rapid increase in the expression of chemokines and genes involved in inflammation. These changes were most pronounced at 1 week post-instillation with Uf-NiO. The expression of Mmp12 mRNA, encoding macrophage metalloelastase 12, was strongly induced immediately following intratracheal instillation. However, expression returned to control levels by 6 months post-instillation expression of various other genes categorized into the detection of chemical stimulus were increased at this time point, at which time the inflammatory response had diminished. These results suggest that residual Uf-NiO in the lungs subacutely initiated distinct cellular events through signal transduction after resolution of the inflammatory response. We conclude that gene expression analysis using DNA microarrays can be extremely useful in assessing the influence of utrafine particles on biological systems.

Closed-loop analysis of cardiovascular variability in rats under restraint stress

Causal transfer function analysis was applied to the heart rate variability and blood pressure variability in normotensive male Sprague-Dawley rats those were measured before, during, and after acute restraint stress. The causal transfer gain (CTG) from systolic blood pressure (SBP) to RR interval (RRI) and CTG from RRI to SBP were estimated. The mean value of the CTG from SBP to RRI in the low-frequency (LF) band (0.27-0.74 Hz) was significantly lower during the restraint period than during the baseline period and remained significantly lower during the recovery period. The mean value of the CTG from RRI to SBP in the LF band, in contrast, was significantly higher during the restraint period than during the baseline period, and during the recovery period it tended to return relatively rapidly to the baseline level. Arterial pressure tended to increase only at the onset of the restraint stress and then to decline not only during the restraint period but also during the recovery period, finally leveling off significantly below the baseline level. These results suggest that restraint stress suppresses the baroreflex control gain not only during the restraint period but also during the recovery period and that this results in the arterial hypotension during the recovery period.