Koichi Sakurai

Inhibition of microsomal lipid peroxidation by baicalein: A possible formation of an iron‐baicalein complex

Baicalein decreased the production of thiobarbituric acid reactive substances, the rate of oxygen consumption and iron reduction in the reaction system of ascorbic acid with FeCl3. Superoxide dismutase, catalase and hydroxyl radical scavengers had no significant effect. Ironchelators had an inhibitory effect similar to that of baicalein. The production of thiobarbituric acid reactive substances of baicalein‐treated microsomes obtained by centrifugation after incubation with baicalein was not observed in the reaction system, but was stimulated by adding iron with increases in concentration. The amount of bound iron to microsomal membranes increased by increasing both the concentration of baicalein and iron. The amount of baicalein bound to microsomal membranes increased with increasing concentration of added baicalein. These results suggest that baicalein bound to microsomal membranes inhibits lipid peroxidation by formating an iron‐baicalein complex.

Requirement of intracellular free thiols for hydrogen peroxide‐induced hypertrophy in cardiomyocytes

Reactive oxygen species (ROS) are by‐products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H2O2 produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H2O2 on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H2O2 induced hypertrophy in control cells in a time‐dependent manner. Pre‐incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H2O2 as compared to non‐BSO pre‐incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre‐incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H2O2, although total thiol levels were not markedly decreased. In the cells pre‐incubated with BSO, hypertrophy was not observed by the addition of H2O2 at any level of concentration. N‐acetyl‐l‐cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H2O2 but also induced hypertrophy in cells pre‐incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H2O2. On the other hand, the hypertrophied cells did not become larger by adding H2O2, but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress. J. Cell. Biochem. 89: 944–955, 2003.

Necrotic pathway in human osteosarcoma Saos-2 cell death induced by chloroacetaldehyde.

Chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, may be responsible for serious adverse effects like encephalopathy in ifosfamide chemotherapy. In this study, we demonstrate that chloroacetaldehyde, but not ifosfamide, induces cell death in human osteosarcoma Saos-2 cells and we investigated the mechanism by which this occurs. Chloroacetaldehyde above 30 μmol/l induced significant cell death in a time-dependent manner. Thiol compounds such as N-acetyl cysteine, glutathione and dithiothreitol protected the cells against chloroacetaldehyde-induced cell death, although other nonthiol compounds and the antioxidative enzymes superoxide dismutase and catalase did not, suggesting that reactive oxygen species might not mediate cell death. In cells exposed to chloroacetaldehyde, levels of both total thiols and glutathione were significantly reduced. Chloroacetaldehyde also collapsed the mitochondrial membrane potential of these cells, induced the release of cytochrome c from mitochondria to the cytosol and significantly reduced cellular ATP levels during the course of death. The mitochondrial potential collapse was also prevented by thiol compounds. Flow cytometric analyses by means of annexin-V and propidium iodide double staining and immunofluorescence staining of active caspase-3 revealed that cells subjected to a lethal dose of chloroacetaldehyde displayed features characteristic of necrosis and that caspase-3 was not activated in response to chloroacetaldehyde. Taken together, these findings suggest that Saos-2 cells exposed to chloroacetaldehyde die by necrosis resulting from a decrease in intracellular thiols, disruption of the mitochondrial membrane potential and the depletion of cellular ATP.

Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937

 Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

Increase of Anti-oxidative Capacity during Differentiation of 3T3-L1 Preadipocytes into Adipocytes

Cells have developed ingenious defense mechanisms in response to oxidative stress. Here, we evaluated changes in anti-oxidative capacity during differentiation of 3T3-L1 preadipocytes into adipocytes. When 3T3-L1 preadipocytes were treated with H2O2 (0.10-2.0 mM) for 21 h, cell viability decreased in response to H2O2 concentration, with an LD50 of approximately 0.35 mM H2O2. In the cells undergoing differentiation at 2 and 6 d, LD50 increased to 1.0 and >2.0 mM H2O2, respectively. These results indicate that resistance to oxidative stress dramatically increased with progression of differentiation of preadipocytes into adipocytes. Catalase activity and GSH content increased in the differentiated cells at 6 d, whereas superoxide dismutase and glutathione peroxidase activities were slightly lower in adipocytes than in preadipocytes. Moreover, knockdown of catalase or depletion of intracellular GSH enhanced the sensitivity to H2O2. When GSH was added to the cells depleted of intracellular GSH, the antioxidant capacity recovered. Autophagy was increased in differentiated adipocytes but was not affected by H2O2 treatment. Therefore, these results suggest that the increase in intracellular catalase activity and GSH content played a role in the increased anti-oxidative capacity of differentiated 3T3-L1 adipocytes.