Significance of Singlet Oxygen Molecule in Pathologies.

Reactive oxygen species, including singlet oxygen, play an important role in the onset and progression of disease, as well as in aging. Singlet oxygen can be formed non-enzymatically by chemical, photochemical, and electron transfer reactions, or as a byproduct of endogenous enzymatic reactions in phagocytosis during inflammation. The imbalance of antioxidant enzymes and antioxidant networks with the generation of singlet oxygen increases oxidative stress, resulting in the undesirable oxidation and modification of biomolecules, such as proteins, DNA, and lipids. This review describes the molecular mechanisms of singlet oxygen production in vivo and methods for the evaluation of damage induced by singlet oxygen. The involvement of singlet oxygen in the pathogenesis of skin and eye diseases is also discussed from the biomolecular perspective. We also present our findings on lipid oxidation products derived from singlet oxygen-mediated oxidation in glaucoma, early diabetes patients, and a mouse model of bronchial asthma. Even in these diseases, oxidation products due to singlet oxygen have not been measured clinically. This review discusses their potential as biomarkers for diagnosis. Recent developments in singlet oxygen scavengers such as carotenoids, which can be utilized to prevent the onset and progression of disease, are also described.

Application of regulation of reactive oxygen species and lipid peroxidation to disease treatment.

Although many diseases in which reactive oxygen species (ROS) and free radicals are involved in their pathogenesis are known, and antioxidants that effectively capture ROS have been identified and developed, there are only a few diseases for which antioxidants have been used for treatment. Here, we discuss on the following four concepts regarding the development of applications for disease treatment by regulating ROS, free radicals, and lipid oxidation with the findings of our research and previous reports. Concept 1) Utilization of antioxidants for disease treatment. In particular, the importance of the timing of starting antioxidant will be discussed. Concept 2) Therapeutic strategies using ROS and free radicals. Methods of inducing ferroptosis, which has been advocated as an iron-dependent cell death, are mentioned. Concept 3) Treatment with drugs that inhibit the synthesis of lipid mediators. In addition to the reduction of inflammatory lipid mediators by inhibiting cyclooxygenase and leukotriene synthesis, we will introduce the possibility of disease treatment with lipoxygenase inhibitors. Concept 4) Disease treatment by inducing the production of useful lipid mediators for disease control. We describe the treatment of inflammatory diseases utilizing pro-resolving mediators and propose potential compounds that activate lipoxygenase to produce these beneficial mediators.

Development of a method for evaluating the mRNA transcription activity of influenza virus RNA-dependent RNA polymerase through real-time reverse transcription polymerase chain reaction.

BACKGROUND: The development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as 32P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the mRNA transcription activity of RdRp without using ultracentrifugation and radioisotopes. RESULTS: RdRp was extracted from viral particles that were purified from the culture supernatant using anionic polymer-coated magnetic beads that can concentrate influenza virus particles from the culture supernatant in approximately 30 min. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on reverse transcription using tagged primers. RT primers were designed to bind to a sequence near the 3' end of mRNA containing a poly A tail for specific recognition of the mRNA, with an 18-nucleotide tag attached to the 5' end of the sequence. The RT reaction was performed with this tagged RT primer, and the amount of mRNA was analyzed using real-time qPCR. Real-time qPCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg2+ concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors. CONCLUSION: This method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral mRNA transcription activity of RdRp.

Induction of a 5-lipoxygenase product by daidzein is involved in the regulation of influenza virus replication.

This study was conducted to evaluate the regulation mechanism of influenza virus replication following treatment of Madin-Darby canine kidney cells with the soy isoflavone daidzein. We performed comparative qualitative and quantitative analyses of lipid peroxide between mock-infected and virus-infected cells treated with or without daidzein, as it had been reported that daidzein was an antioxidant and lipid peroxide levels increased upon virus infection. Contrary to our belief, lipid peroxides were not elevated in virus-infected cells and no decrease in lipid peroxides was observed in daidzein-treated cells. In daidzein-treated cells, 5-hydroxyeicosatetraenoic acid, the 5-lipoxygenase product derived from arachidonate, was significantly elevated compared to other lipid peroxides. Zileuton (5-lipoxygenase inhibitor) and 5-lipoxygenase knockdown reduced the daidzein-induced antiviral effect. Moreover, virus replication was regulated by treatment with 5-hydroperoxyeicosatetraenoic acid, a precursor of 5-hydroxyeicosatetraenoic acid and 5-lipoxygenase primary product. These results suggest that daidzein regulates virus replication via signal transduction through 5-lipoxygenase products.

Probucol induces the generation of lipid peroxidation products in erythrocytes and plasma of male cynomolgus macaques.

We previously reported that probucol, a lipid lowering agent, protected mice from malaria infection via depletion in plasma α-tocopherol. The antioxidant α-tocopherol in host circulation is necessary for the malaria parasites to protect themselves from oxidative stress in erythrocytes where high amounts of reactive oxygen species are generated. To assess the potential for the clinical application of probucol as an anti-malarial therapy, it was necessary to determine the effects of probucol by using primate experiments. Here we verified that probucol induces an α-tocopherol decrement in cynomolgus macaque erythrocytes and plasma. After 2 weeks of probucol administration at doses of 200 or 400 mg/kg/day, the α-tocopherol contents in erythrocytes tended to decrease. The contents of hydroxyoctadecadienoic acids and 7ß-hydroxycholesterol, peroxidation products derived from linoleic acid and cholesterol, respectively, increased in erythrocytes. On the other hand, plasma α-tocopherol concentration showed a marginal decrement. Plasma lipid peroxidation products were transiently increased in the early stages of probucol administration. No adverse effects were observed throughout the experiment, although the dosage of probucol was higher than the clinical maximum dosage. Considering that malaria proliferates in erythrocytes, probucol-induced disruption of redox homeostasis in erythrocytes could be effective in the inhibition of parasite proliferation.

Ascorbic acid insufficiency impairs spatial memory formation in juvenile AKR1A-knockout mice.

AKR1A, an aldo-keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A-/- mice produce considerably less ascorbic acid (about 10%) compared to AKR1A+/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A-/- male mice were weaned at 4 weeks of age and groups that received ascorbic acid supplementation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A-/- mice that received no supplementation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A-/- mice at day 7 after weaning. To the contrary, the young adult AKR1A-/- mice at 13-15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A+/+ mice or ascorbic acid-supplemented AKR1A-/- mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.

Early diagnosis of type 2 diabetes based on multiple biomarkers and non-invasive indices.

We previously reported that type 2 diabetes risk, early impaired glucose tolerance and insulin resistance can be predicted by measuring the fasting levels of certain biomarkers. Here we validated these findings in randomly recruited healthy volunteers (n = 101) based on biomarker expression as well as various non-invasive indices. Weight, body mass index, waist circumference and visceral fat differed between individuals with impaired fasting glucose and/or impaired glucose tolerance, and normal subjects. Fasting plasma levels of glycated hemoglobin, leptin, pro-insulin and retinol binding protein 4 differed between impaired fasting glucose/impaired glucose tolerance and normal subjects group and between newly detected diabetes and normal subjects group. Insulin resistance was correlated with fasting levels of insulin and leptin/adiponectin (r = 0.913); of insulin, retinol binding protein 4 and leptin/adiponectin (r = 0.903); and of insulin, glycated albumin, and leptin/adiponectin (r = 0.913). Type 2 diabetes risk, early impaired glucose tolerance and insulin resistance were predicted with >98% specificity and sensitivity by comparing fasting glucose levels to the estimated Matsuda Index based on fasting levels of insulin, adiponectin and leptin with or without oxidative lineolate metabolites. Non-invasive indices are slightly correlated with glucose tolerance and insulin resistance but do not increase the accuracy of predicting type 2 diabetes risk.

An open sandwich immunoassay for detection of 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid.

Lipid peroxidation is involved in many disorders and diseases such as cardiovascular disease, cancers, neurodegenerative diseases, and even aging. Lipid peroxidation products existing in blood or bodily fluids are very important biomarkers for the diagnosis of such diseases. In particular, 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid (13-(E,E)-HODE) is an oxidiation product of linoleic acid, which is an important biomarker for many diseases such as diabetes and Alzheimer's disease. In this study, we successfully displayed the antigen-binding fragment of an antibody produced by hybridoma 1213-1 on the M13 phage and performed analysis of the antibody variable region genes. The blast results suggested that it is a novel antibody. We also developed a phage-antibody-based competitive ELISA and a novel Open Sandwich ELISA (OS ELISA) for the detection of 13-(E,E)-HODE. The OS ELISA showed a limit of detection (LOD) of 15.6 nM of 13-(E,E)-HODE and low cross-reactivity with other HODE such as 9-(E,E)-HODE. Another format of the open sandwich ELISA with purified maltose binding protein-fused VL and VH-phage showed a lower LOD of 2.2 nM of 13-(E,E)-HODE, which may be sensitive enough to detect the concentration of 13-(E,E)-HODE in patients' blood samples. This is the first OS ELISA for the detection of lipids, and we believe it also represents the first molecular cloning of anti-HODE antibody genes.

Probucol dramatically enhances dihydroartemisinin effect in murine malaria.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been adopted as national policy for the first-line treatment in large number of malaria-endemic regions. However, artemisinin-resistant parasites have emerged and are spreading, with slow-cleaning parasites being reported in patients treated with ACT. It means that more parasites are exposed to the partner drug alone and the risk of developing resistant parasites against the partner drug is increasing. Therefore, the development of a new method to enhance the effect of artemisinin is required. In this study, the potential effect of probucol as a combination drug of dihydroartemisinin (DHA), an artemisinin derivative, was examined. METHODS: C57BL/6 J mice infected with Plasmodium yoelii XL-17 were treated with probucol and/or DHA. The mice were fed with a probucol mixed diet from 2 weeks before infection and through infection period. DHA was injected to mice three to 5 days post infection once a day. In addition, 0.5 % (w/w) probucol was mixed with vitamin E supplemented diet (800 mg/kg) and fed to mice infected with P. yoelii XL-17 to examine the mechanisms of probucol on murine malaria. Furthermore, 8-OHdG, a biomarker of oxidized DNA, was detected in infected red blood cells (iRBC) taken from infected mice by immunofluorescent staining. RESULTS: With dose-dependent manner, both probucol and DHA decreased parasitaemia and increased survival rate of mice infected with P. yoelii XL-17. A significantly larger amount of 8-OHdG was detected in iRBC taking from probucol-treated mice than control mice. In addition, a large amount of vitamin E supplementation eliminated the effect of probucol against P. yoelii XL-17 infection and lowered the effect of probucol on host plasma vitamin E concentration. The effective doses for probucol and DHA were 0.5 % and 30 mg/kg, respectively, in each single treatment. While the combination treatment of 0.25 % probucol and 7.5 mg/kg DHA was effective in all mice from P. yoelii XL-17 infection. CONCLUSION: This study demonstrated that probucol has some impact on malaria by oxidative stress through the induction of host plasma vitamin E deficiency. Moreover, the effective dose of DHA on malaria was decreased by prophylactic treatment of probucol. This finding indicates that probucol might be a candidate for a prophylactic treatment drug to enhance the effect of DHA.

Trichloroethylene exposure aggravates behavioral abnormalities in mice that are deficient in superoxide dismutase.

Trichloroethylene (TCE) has been implicated as a causative agent for Parkinson's disease (PD). The administration of TCE to rodents induces neurotoxicity associated with dopaminergic neuron death, and evidence suggests that oxidative stress as a major player in the progression of PD. Here we report on TCE-induced behavioral abnormality in mice that are deficient in superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-deficient (Sod1(-/-)) mice were intraperitoneally administered TCE (500 mg/kg) over a period of 4 weeks. Although the TCE-administrated Sod1(-/-) mice showed marked abnormal motor behavior, no significant differences were observed among the experimental groups by biochemical and histopathological analyses. However, treating mouse neuroblastoma-derived NB2a cells with TCE resulted in the down regulation of the SOD1 protein and elevated oxidative stress under conditions where SOD1 production was suppressed. Taken together, these data indicate that SOD1 plays a pivotal role in protecting motor neuron function against TCE toxicity.

Dynamics of hydroxyoctadecadienoic acid in epilepsy patients treated with valproic acid.

BACKGROUND: The effects of valproic acid (VPA) on oxidative stress are controversial due to differences in experimental conditions. Recently, total hydroxyoctadecadienoic acid (tHODE), derived from linoleic acid, was proposed as a potent biomarker to evaluate oxidative stress. METHODS: The subjects consisted of 10 new-onset epilepsy patients treated with VPA. We measured plasma tHODE consecutively for 1 year to evaluate the degree of oxidative stress and then compared plasma tHODE at the beginning and the end of experiments with the peak level. Ten age-matched healthy subjects were recruited as a control group and their plasma tHODE was compared to the initial plasma tHODE of the epilepsy group. Measurements were done using liquid chromatography-tandem mass spectrometry. RESULTS: Mean initial plasma tHODE in the epilepsy group was 165.2 ± 76.8 nmol/L, which was not significantly different from that of the control group (199.3 ± 62.5 nmol/L). In five epilepsy patients, plasma tHODE increased above the pathological level in 6 months, but returned to normal within 1 year. In the whole group, the difference plasma tHODE between peak, at the beginning and 1 year later, was significant. CONCLUSION: Oxidative stress generated by VPA increased temporarily, but decreased to normal after 1 year. it is reasonable to be concerned about the effects of oxidative stress, especially at the start of VPA treatment.

Effect of anti-hyperlipidemia drugs on the alpha-tocopherol concentration and their potential for murine malaria infection.

The current preventions of malaria are protection against mosquito bites and taking chemoprophylactic anti-malarial drugs. However, drug therapies are usually associated with adverse events and emergency of drug-resistant malaria parasites. Previous study showed that host plasma alpha-tocopherol deficiency enhanced resistance against malaria infection in mice. Here, we report a new prevention strategy against malaria by using anti-hyperlipidemia drugs, ezetimibe, berberine, cholestyramine, and probucol to modify the host plasma alpha-tocopherol concentration. The drugs were mixed with diet and fed to C57BL/6J mice for 2 weeks. Although all drugs reduced plasma alpha-tocopherol concentration after 2 weeks of feeding, probucol-treated mice showed 90 % reduction and it was the lowest alpha-tocopherol concentration among the four drugs. Ezetimibe, berberine, and combination of ezetimibe and berberine pretreatment for 2 weeks were not effective against infection of Plasmodium yoelii XL17, a lethal strain, for survival and parasitemia in mice. Two-week pretreatment and 1-week treatment after infection of cholestyramine had also no effect on malaria infection. Survival rates of cholestyramine, ezetimibe, and/or berberine treated mice were 0-22 %. However, probucol caused significant decrease in parasitemia and increased in mice survival following 2-week pretreatment and 1-week treatment after infection. All control mice died while all probucol treated mice survived during the course of infection. Thus, probucol which reduced plasma alpha-tocopherol concentration was effective in enhancing the host to resist malaria infection in mice. Our finding indicates that plasma alpha-tocopherol reducing drugs like probucol might be a candidate for beneficial prevention strategy for travelers from malaria-free area.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

Enhancement of lipid peroxidation and its amelioration by vitamin E in a subject with mutations in the SBP2 gene.

Selenocysteine (Sec) insertion sequence-binding protein 2 (SBP2) is essential for the biosynthesis of Sec-containing proteins, termed selenoproteins. Subjects with mutations in the SBP2 gene have decreased levels of several selenoproteins, resulting in a complex phenotype. Selenoproteins play a significant role in antioxidative defense, and deficiencies in these proteins can lead to increased oxidative stress. However, lipid peroxidation and the effects of antioxidants in subjects with SBP2 gene mutations have not been studied. In the present study, we evaluated the lipid peroxidation products in the blood of a subject (the proband) with mutations in the SBP2 gene. We found that the proband had higher levels of free radical-mediated lipid peroxidation products, such as 7ß-hydroxycholesterol, than the control subjects. Treatment of the proband with vitamin E (α-tocopherol acetate, 100 mg/day), a lipid-soluble antioxidant, for 2 years reduced lipid peroxidation product levels to those of control subjects. Withdrawal of vitamin E treatment for 7 months resulted in an increase in lipid peroxidation products. Collectively, these results clearly indicate that free radical-mediated oxidative stress is increased in the subject with SBP2 gene mutations and that vitamin E treatment effectively inhibits the generation of lipid peroxidation products.

Photothermic regulation of gene expression triggered by laser-induced carbon nanohorns.

The development of optical methods to control cellular functions is important for various biological applications. In particular, heat shock promoter-mediated gene expression systems by laser light are attractive targets for controlling cellular functions. However, previous approaches have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, which have poor penetration into biological tissue. Biological tissue is relatively transparent to light inside the diagnostic window at wavelengths of 650-1,100 nm. Here we present a unique optical biotechnological method using carbon nanohorn (CNH) that transforms energy from diagnostic window laser light to heat to control the expression of various genes. We report that with this method, laser irradiation within the diagnostic window resulted in effective heat generation and thus caused heat shock promoter-mediated gene expression. This study provides an important step forward in the development of light-manipulated gene expression technologies.

The role of lipid peroxidation in neurological disorders.

There has been much evidence demonstrating the involvement of oxidative stress in the pathology of neurological disorders. Moreover, the vulnerability of the central nervous system to reactive oxygen species mediated injury is well established since neurons consume large amounts of oxygen, the brain has many areas containing high iron content, and neuronal mitochondria generate large amounts of hydrogen peroxide. Furthermore, neuronal membranes are rich in polyunsaturated fatty acids, which are particularly susceptible to oxidative stress. Recently, the biological roles of products produced by lipid peroxidation have received much attention, not only for their pathological mechanisms associated with neurological disorders, but also for their practical clinical applications as biomarkers. Here, we discuss the production mechanisms of reactive oxygen species in some neurological disorders, including Alzheimer's disease, Down syndrome, Parkinson's disease, and stroke. We also describe lipid peroxidation biomarkers for evaluating oxidative stress.

Type 2 diabetes model TSOD mouse is exposed to oxidative stress at young age.

Tsumura Suzuki Obese Diabetes (TSOD) mouse, a model of obese type 2 diabetes, older than around 11 weeks of age develops diabetic phenotypes. Previous studies have indicated that the development of diabetes is partly due to three loci associated with body weight and glucose homeostasis. However, little is known about the initial events triggering the development of the diabetic phenotypes in TSOD mouse. Here, we investigated the alteration of diabetes-related parameters, including the levels of blood glucose and inflammatory cytokines, and the oxidative stress status, in young TSOD mice. TSOD mice at 5 weeks of age showed increases in body weight and plasma total cholesterol level, but not hyperglycemia or impaired glucose tolerance, compared with age-matched control Tsumura Suzuki Non-Obese (TSNO) mice. Plasma tumor necrosis factor (TNF)-α and interleukin (IL)-6 were not detected in TSOD mice at 5 weeks of age. However, plasma total hydroxyoctadecadienoic acid (tHODE), a biomarker of oxidative stress, was increased in TSOD mice relative to TSNO mice at same age. The results demonstrated that young TSOD mice are exposed to oxidative stress before developing the diabetic phenotypes, and suggested that oxidative stress is an initial event triggering the development of diabetes in TSOD mice.

DHA concentration of red blood cells is inversely associated with markers of lipid peroxidation in men taking DHA supplement.

An increase in the proportion of fatty acids with higher numbers of double bonds is believed to increase lipid peroxidation, which augments the risk for many chronic diseases. (n-3) Polyunsaturated fatty acids provide various health benefits, but there is a concern that they might increase lipid peroxidation. We examined the effects of docosahexaenoic acid [22:6 (n-3)] supplementation on lipid peroxidation markers in plasma and red blood cells (RBC) and their associations with red blood cell and plasma fatty acids. Hypertriglyceridemic men (n = 17 per group) aged 39-66 years participated in a double-blind, randomized, placebo-controlled, parallel study. They received no supplements for the first 8 days and then received 7.5 g/day docosahexaenoic acid oil (3 g/day docosahexaenoic acid) or olive oil (placebo) for 90 days. Fasting blood samples were collected 0, 45, and 91 days after supplementation. Docosahexaenoic acid supplementation did not change plasma or RBC concentrations of lipid peroxidation markers (total hydroxyoctadecadienoic acid, total hydroxyeicosatetraenoic acid, total 8-isoprostaglandin F2α, 7α-hydroxycholesterol, 7ß-hydroxycholesterol) when pre- and post-supplement values were compared. However, the post-supplement docosahexaenoic acid (DHA) concentration was inversely associated with RBC concentrations of ZE-HODE, EE-HODE, t-HODE, and total 8-isoprostaglandin F2α, (p<0.05). RBC concentration of hydroxycholesterol was also inversely associated with DHA but it did not attain significance (p = 0.07). Our results suggest that increased concentration of DHA in RBC lipids reduced lipid peroxidation. This may be another health benefit of DHA in addition to its many other health promoting effects.

A novel role for α-tocopherol transfer protein (α-TTP) in protecting against chloroquine toxicity.

Chloroquine (CQ) is a widely prescribed anti-malarial agent and is also prescribed to treat autoimmune diseases. Clinical treatment with CQ is often accompanied by serious side effects such as hepatitis and retinopathy. As a weak base, CQ accumulates in intracellular acidic organelles, raises the pH, and induces osmotic swelling and permeabilization of acidic organelles, which account for CQ-induced cytotoxicity. We reported previously that CQ treatment caused α-tocopherol transfer protein (α-TTP), a gene product of familial vitamin E deficiency, to change its location from the cytosol to the surface of acidic organelles. Here we show that α-TTP plays a novel role in protecting against CQ toxicity both in vitro and in vivo. In the presence of CQ, rat hepatoma McARH7777 cells, which do not express α-TTP endogenously, showed more severe cytotoxicity, such as larger vacuolation of acidic organelles and caspase activation, than α-TTP transfectant cells. Similarly, α-TTP knockout mice showed more severe CQ toxicity, such as hepatotoxicity and retinopathy, than wild-type mice. These effects were not ameliorated by vitamin E supplementation. In contrast to bafilomycin A1 treatment, which prevents CQ accumulation in cells by raising the pH of acidic organelles, α-TTP expression prevented CQ accumulation without affecting the pH of acidic organelles. Taken together, our data suggest that α-TTP protects against CQ toxicity by preventing CQ accumulation in acidic organelles through a mechanism distinct from vitamin E transport.

Lipid peroxidation biomarkers for evaluating oxidative stress and assessing antioxidant capacity in vivo.

Recently, the biological roles of lipid peroxidation products have received a great deal of attention not only for elucidating pathological mechanisms but also for practical clinical applications as biomarkers. In the last 50 years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as major primary products, but they are substrates for various enzymes and they also undergo various secondary reactions. During this decade, hydroxyoctadecadienoic acid from linoleates, F(2)-isoprostanes from arachidonates, and neuroprostanes from docosahexanoates have been proposed as biomarkers for evaluating oxidative stress in vivo and its related diseases. The implications of lipid peroxidation products in vivo will be briefly reviewed and their practical applications will be discussed.