Shosuke Kojo

Vitamin C: Basic Metabolism and Its Function as an Index of Oxidative Stress

Vitamin C (ASC) is well known as an outstanding antioxidant in animal tissues. This concept is reviewed from a chemical standpoint, starting from a chemical view of radical reactions in the cell. ASC, vitamin E, and lipid hydroperoxide were selected as key molecules involved in radical reactions in the cell, and their efficiencies as an index of oxidative stress were evaluated. At first, methods for specific and sensitive determination of ASC and lipid hydroperoxide were developed. Based on comparisons of these indices during oxidative stress in typical pathological conditions, such as diabetes and liver damage by toxicants, ASC concentration was found to be the most sensitive index in animal tissues. Antioxidative effect of food factors in vivo can be evaluated on the basis of these indices. Analysis of oxidation of low-density lipoprotein (LDL) revealed that degradation and cross-link of apolipoprotein B-100 (apoB) are extremely facile processes. Fragmented and conjugated apoB proteins are present in normal human serum, and tend to increase with age based on immunoblot analysis. Estimation of these products allows us a mechanism-based diagnosis of atherosclerosis. A significant relationship between plasma ASC level and the sum of these apoB products was found. In conclusion, specifically determined ASC concentration sensitively reflects oxidative stress in tissues.

Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver

After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.

Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species

Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2′,7′‐dichlorodihydrofluorescin diacetate (DCFH‐DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time‐ and dose‐dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase‐1, a stress‐induced protein, in hemin‐treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin‐synthesizing erythroleukemia K562 cells. When K562 cells were treated with δ‐aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH‐DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.

Evaluation of oxidative stress during apoptosis and necrosis caused by d-galactosamine in rat liver

Eighteen and twenty-four hours after intraperitoneal administration of D-galactosamine (1g/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared with that in the control group given saline. Histological examinations including the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method found apoptotic hepatocytes 18 hr after the administration of D-galactosamine. Caspase-3 activity was barely detectable in the plasma of control rats, but increased significantly 24 hr after drug administration along with a dramatic increase in glutamate-oxaloacetate transaminase (GOT). These results indicated that D-galactosamine causes apoptosis in the liver by activating caspase-3, which is released to the plasma by secondary necrosis. The concentration of lipid hydroperoxides in the liver increased significantly 24 hr after D-galactosamine administration. In contrast, the concentration of vitamin C in the liver decreased significantly 18 and 24 hr after D-galactosamine administration. These results suggest that D-galactosamine induces severe oxidative stress in the liver, leading to extensive necrosis.

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMS Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype. METHODS AND RESULTS SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice. CONCLUSION These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver

After 12 h of thioacetamide (500 mg/kg body weight) administration to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group. In plasma, the activity of caspase-3 was barely detectable in the control rat, but had increased significantly after 24 h of drug administration along with a dramatic increase in GOT. These results indicate that thioacetamide causes apoptosis in the liver by activating caspase-3, which is released to plasma by successive necrosis. At 24 h, the concentration of liver lipid hydroperoxides, a mediator of radical reaction, was 2.2 times as high as that of control rats. After 12 and 24 h of thioacetamide administration, the liver concentrations of vitamins C and E decreased significantly. The decrease of antioxidants and formation of lipid hydroperoxides 24 h after thioacetamide administration support the view that extensive radical reactions occur in the liver during the necrotic process.

Effects of chronic acetyl-L-carnitine treatment on brain lipid hydroperoxide level and passive avoidance learning in senescence-accelerated mice.

In the present study, we examined the effects of acetyl-L-carnitine (ALC) on the brain lipid hydroperoxide level and on passive avoidance performance in senescence-acceleration-prone 8 mice (SAMP8). Mice were treated intraperitoneally with either saline or ALC (100 or 400 mg/kg) three times a week up to 4 months of age (starting at 3 weeks of age). In 4-month-old SAMP8, the deficit in learning and memory seen in saline-treated controls was significantly ameliorated in 400 mg/kg ALC-treated SAMP8, and the brain lipid hydroperoxide level was significantly lower in the 400 mg/kg ALC-treated group than in the saline-treated controls. Administration of 100 mg/kg ALC to SAMP8 did not have significant effect on learning and memory performance or on the brain lipid hydroperoxide level (by comparison with the saline-treated controls). These results suggest that ALC has antioxidant activity towards oxidative stress, and that the improvement in cognitive ability seen with ALC may occur through an amelioration of cellular dysfunction via an inhibition of the increase in lipid hydroperoxidation observed in the brain tissue of untreated SAMP8.

Involvement of hydrogen peroxide and hydroxyl radical in chemically induced apoptosis of HL-60 cells

Effects of three kinds of antagonists against reactive oxygen species were evaluated at the same time in chemically induced apoptosis of human leukemic HL-60 cells. Apoptosis of HL-60 cells induced by actinomycin D, H7, 1-beta-D-arabinofuranosylcytosine, and daunorubicin was inhibited significantly by radical scavengers (vitamin E, N-acetyl-L-cysteine, and mercaptoethanol), catalase, and a spin trap, N-t-butyl-alpha-phenylnitrone. These results suggest that hydrogen peroxide and hydroxyl radical are common mediators of apoptosis caused by these chemicals with apparently different functional mechanisms. The consumption of vitamin E to inhibit apoptosis induced by actinomycin D was undetectable, suggesting that the generation of reactive oxygen species during apoptosis was not very extensive. Radicals were suggested to be a mediator of apoptosis of HL-60 cells induced by cisplatin based on the observations that the above inhibitors, except catalase, effectively inhibited apoptosis by the drug.

Association of ceramides in human plasma with risk factors of atherosclerosis

Atherosclerosis is a multifactorial disorder. Recent studies indicate that the plasma level of sphingomyelin, which yields ceramide, correlates with the risk of coronary heart disease. Therefore, ceramide, a well-known lipid causing apoptosis in various cell types, may contribute to atherogenesis. We examined the relationship between ceramide concentration and risk factors of atherosclerosis in normal human plasma using electrospray tandem mass spectrometry (LC-MS/MS). Major ceramides in human plasma were C24∶0 and C24∶1. The ceramide concentration showed a significant positive correlation with total cholesterol (TC) and triglycerides (TG). In addition, plasma ceramide level increased drastically at a high level of LDL cholesterol (more than 170 mg/dL). Our previous studies demonstrated that the sum of fragmented and conjugated apolipoprotein B-100 proteins (B-ox), which were products of a radical reaction of LDL as well as plasma, was a reliable index of atherosclerosis. B-ox showed a significant positive correlation with the plasma ceramide level. Based on these results, we propose that the ceramide level in human plasma is a risk factor at the early stages of atherosclerosis.

Age-dependent changes in lipid peroxide levels in peripheral organs, but not in brain, in senescence-accelerated mice

The tissue concentration of lipid peroxides was determined in the brain, heart, liver, lung and kidney of accelerated senescence-prone (SAMP-8) and -resistant (SAMR-1) mice at 3, 6 and 9 months of age by a method involving chemical derivatization and high performance liquid chromatography. The level of lipid peroxides in the brain did not show an age-dependent change, but at each age the brain level of lipid peroxides was significantly higher in SAMP-8 than in SAMR-1. In contrast, the lipid peroxide levels in the peripheral organs showed increases with aging in both strains, and they were significantly higher in SAMP-8 than in SAMR-1 at both 3 and 6 months of age (except at 3 months of age in the kidney). These results suggest that increased oxidative stress in the brain and peripheral organs is a cause of the senescence-related degeneration and impairments seen in SAMP-8.