Sadako Tokumaru

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.

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.

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.

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.

Specific and sensitive determination of lipid hydroperoxides with chemical derivatization into 1-naphthyldiphenylphosphine oxide and high-performance liquid chromatography

Abstract A highly sensitive and specific high-performance liquid chromatographic (HPLC) method to determine the hydroperoxide level in biological samples involving the oxidation of 1-naphthyldiphenylphosphine into its oxide was developed. The detection limit of the method applied to the determination of cumene hydroperoxide was 1 pmol and the yield of the phosphine oxide based on the hydroperoxide in the concentration range from 0.2 μM to 1 mM was 85–100%. The linearity between the amount of the hydroperoxide and the HPLC peak height was excellent in a wide range (1 pmol to 5 nmol). The recoveries of cumene hydroperoxide from saline and homogenates of mouse brain, heart, kidney and liver were 88.5 ± 10, 83.3 ± 13, 75.3 ± 1.0, 46.3 ± 13.4 and 18.7 ± 10%, respectively. The recovery of exogenously added lecithin hydroperoxide from mouse liver homogenate was 52.4 ± 9.8%, while that from saline was 63.6 ± 5.4%. The peroxide levels in the brain, heart, kidney and liver of 5-week-old mice were determined to be 474 ± 170, 426 ± 183, 655 ± 121 and 239 ± 31 pmol/mg protein, respectively.

Possible involvement of radical reactions in desialylation of LDL

The role of oxidatively modified LDL in the pathogenesis of atherosclerosis has been well documented. These studies have focused on modifications of lipid and protein parts of LDL. Recently desialylated LDL has received attention in relation to atherosclerosis and coronary artery disease. We examined the possible involvement of radical reactions in desialylation of LDL. Human LDL was subjected to oxidative damage using Cu2+ ion. As the conjugated dienes monitored by absorption at 234 nm increased, the content of sialic acid decreased steadily. Both the elevation of conjugated diene and the decrease of sialic acid were inhibited by β‐mercaptoethanol, a typical radical scavenger. Besides, both butylated hydroxytoluene and a nitrogen atmosphere inhibited the decrease of sialic acid. These inhibition experiments suggested that sialic acid moieties in LDL were reactive toward radicals.

Increase of Lipid Hydroperoxides in Tissues of Vitamin E-Deficient Rats

The level of lipid hydroperoxides was determined by a newly developed method in rat tissues of vitamin E deficiency, which was a good in vivo model of enhanced radical reactions. In the heart, lung and kidney, the level of lipid hydroperoxides increased significantly as early as 4 weeks after feeding on a tocopherol-deficient diet compared with that of the control group. After 8 weeks of the deficiency, similar results were obtained. These results indicate that the lipid hydroperoxide is available as an extremely sensitive indicator of lipid peroxidation in these organs, because it takes several months to detect manifestations of the vitamin deficiency based on conventional indices.

Change of the lipid hydroperoxide level in mouse organs on ageing

Change in the total level of tissue lipid hydroperoxides during ageing in the mouse was determined by our newly developed specific and sensitive method. The hepatic level of lipid hydroperoxides of 5-week-old mice was 239 +/- 31 pmol/mg protein. Hydroperoxide levels in the liver of 20-, 30-, 40-, 60- and 85-week-old groups were 487 +/- 115, 348 +/- 87, 395 +/- 65, 498 +/- 98 and 431 +/- 81 pmol/mg protein, respectively, and these values were significantly higher than the content of the 5-week-old animals. In the heart and kidney, the level of lipid hydroperoxides increased also significantly at 20 weeks of age compared with that of the 5 week-old mouse. The hydroperoxide level did not increase significantly thereafter until 85 weeks of age. The hydroperoxide level in the brain did not change during 5-85 weeks of age.

Increase of lipid hydroperoxides in liver mitochondria and inhibition of cytochrome oxidase by carbon tetrachloride intoxication in rats.

The cellular localization of lipid hydroperoxides was determined for the first time in mitochondria, microsomes and cytosol of rat liver using a specific method involving chemical derivatization and HPLC. Mitochondria contained the highest level of hydroperoxides. After 6h of intragastric administration of carbon tetrachloride (CCl4) to rats (2 ml/kg body weight), the concentration of lipid hydroperoxides increased significantly in liver mitochondria and cytochrome oxidase activity was inhibited to 35% of the control rats. The mitochondrial content of haem a decreased to 60% of the control at 12h of CCl4 administration. In vitro reaction of mitochondria with CCl4 caused inactivation of cytochrome oxidase. These observations suggested that cytochrome oxidase and haem a in mitochondria were targets of CCl4.