Mechanism of hydrogen peroxide-induced apoptosis of the human osteosarcoma cell line HS-Os-1

In our previous study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Based on these results, the radioresistance of the human osteosarcoma cell line HS-Os-1, was considered to arise, at least in part, from the low level of ROS formation following irradiation, which in turn may have resulted from the strong scavenging ability of the cells for free radicals, including hydroxyl radicals. Therefore, in this study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that addition of 1 or 10 mM hydrogen peroxide induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation is involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells.     

Mechanism of apoptotic resistance of human osteosarcoma cell line,HS-Os-1, against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of Dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1, which was established from an osteoblastic tumor that arose in the left humerus of an 11-year-old girl and was already morphologically characterized in vitro and in vivo. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells; that mitochondrial membrane potential was preserved; and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. Therefore, the origin of the close similarity of radiosensitivity between adult articular chondrocytes and the human osteosarcoma cell line HS-Os-1, is considered to involve the low degree of ROS formation following irradiation; the similarity possibly results from the strong scavenging ability of these two kinds of cells for free radicals including hydroxyl radicals.     

Apoptotic-resistance of the human osteosarcoma cell line HS-Os-1 to irradiation is converted to apoptotic-susceptibility by hydrogen peroxide: a potent role of hydrogen peroxide as a new radiosensitizer

In our previous study, we demonstrated that the radioresistance of the human osteosarcoma cell line HS-Os-1, was considered to arise, at least in part, from the low level of ROS formation following irradiation, which in turn may have resulted from the strong scavenging ability of the cells for free radicals, including hydroxyl radicals. Following the study, we found that addition of 1 or 10 mM hydrogen peroxide induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore speculated that combined use of irradiation and hydrogen peroxide might exert an additive effect for apoptotic-resistant tumors such as the human osteosarcoma cell line HS-Os-1, in terms of preservation of the radiation-induced hydroxyl radical production supported by the intracellular ROS formation that is induced by exogenous hydrogen peroxide addition. Therefore, in this study, we examined the effect of various doses of irradiation on the existence of 0.1 mM hydrogen peroxide in the culture medium. We found that irradiation with 10 or 20 Gy, under the condition of the presence of 0.1 mM hydrogen peroxide, induced ROS formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1, though ROS formation and oxidative DNA damage were scarcely seen in response to irradiation of up to 30 Gy, as was shown in our previous study. We therefore concluded that the combined modality of irradiation and such a low concentration of hydrogen peroxide (0.1 mM) is potentially applicable in clinical radiotherapy for many kinds of apoptotic-resistant neoplasms in terms of achieving both local control and improving survival benefit of patients.     

Comparison of radiation-induced reactive oxygen species formation in adult articular chondrocytes and that in human peripheral T cells: possible implication in radiosensitivity

Previously, we examined the formation of reactive oxygen species (ROS) in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation in adult articular chondrocytes, which were demonstrated to be highly resistant to apoptosis in our previous study. We found that ROS formation was actually scarcely seen after irradiation of up to 20 Gy in these cells. Therefore, the origin of the great difference of radiosensitivity between T lymphocytes and adult articular chondrocytes is considered to lie in the degree of ROS formation following irradiation, with this difference possibly resulting from the scavenging acuity of these two kinds of normal tissue cells for free radicals including hydroxyl radicals.     

Reactive oxygen species-producing site in hydrogen peroxide-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that the addition of 1 or 10 mM hydrogen peroxide induced reactive oxygen species (ROS) formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation was involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells. In contrast to the osteosarcoma cell line HS-Os-1, human peripheral T cells are considered to be easily susceptible to oxidative stress, because these cells lack peroxidase activity. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization, were observed in these cells. Based on the above results, there exists an apoptotic cascade involving early lysosomal membrane destabilization in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis.     

Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.     

Radiation-induced oxidative DNA damage, 8-oxoguanine,in human peripheral T cells

The mechanism leading to the high level of radiosensitivity of T lymphocytes has not yet been fully described. In our previous study, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h after irradiation of 5 Gy, respectively. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c release was confirmed. In order to elucidate the mechanism which occurs prior to the mitochondrial membrane potential changes, we examined in the present study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we concluded that it remains necessary to evaluate the extent of radiation-induced oxidative DNA damage. Furthermore, it is important to analyze superoxide radical production and scavenging in terms of the variety of radiosensitivities found among various types of normal tissue cells and neoplastic cells.     

Reactive oxygen species-producing site in radiation and hydrogen peroxide-induced apoptosis of human peripheral T cells: Involvement of lysosomal membrane destabilization

In our previous studies, we showed that the apoptotic resistance of the human osteosarcoma cell line HS-Os-1 against irradiation was easily converted to a state of apoptotic-susceptibility by the addition of a relatively low concentration of hydrogen peroxide to the culture medium just prior to irradiation. When we consider the combined use of radiotherapy and hydrogen peroxide in a clinical setting for patients with radioresistant neoplasms, we need to be careful of the possible augmentation of the radiation effect to normal tissues of patients who undergo radiation therapy for their tumor in the presence of a low concentration of hydrogen peroxide in their topical tumor tissue. Therefore, we examined the combined effect of irradiation and hydrogen peroxide compared to that of irradiation alone for human peripheral T cells which were considered to be representative of normal tissue susceptible to apoptosis induced by irradiation. In this study, we compared the morphological changes in human peripheral T cells between both groups by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently augmented in the mitochondria and/or lysosomes instead of in the nuclei of irradiated T cells in the presence of a low concentration of hydrogen peroxide compared to those treated with irradiation alone. Moreover, dysfunction of mitochondrial membrane potential was also more evidently shown in human peripheral T cells irradiated under existence of a low concentration of hydrogen peroxide compared to T cells treated with 5 Gy irradiation alone. Based on these results, we concluded the possible existence of an augmentation effect of irradiation by the existence of a low concentration of hydrogen peroxide for human peripheral T cells. Therefore, we should be alert for the combined effects of radiation therapy and hydrogen peroxide on normal tissues in possible clinical situations when this combination is used for treatment of patients having radioresistant neoplasms such as osteosarcoma, malignant melanoma, and glioblastoma multiforme.     

Impact of reactive oxygen species on spontaneous mutagenesis in Escherichia coli

Reactive oxygen species (ROS) are potent oxidants that attack chromosomal DNA and free nucleotides, leading to oxidative DNA damage that causes genetic alterations. To avoid the ROS-mediated mutagenesis, cells have elaborate mechanisms including powerful antioxidant components and repair pathways that eliminate oxidative DNA damage. Because of the effective anti-mutagenic functions, it has been unclear to what extent the ROS contribute to spontaneous mutagenesis. Here we show that a significant portion of spontaneous mutations is actually caused by the ROS in aerobically growing Escherichia coli cells. Using the rpsL gene as a mutational target sequence, we established an experimental procedure to analyze spontaneous mutations occurring under a strictly anaerobic condition. Strong mutator phenotypes of cells defective in both mutM and mutY genes or ones lacking mutT gene were completely suppressed under the anaerobic condition, indicative of an absence of hydroxyl radicals in the cells. From a series of analyses with wild-type E. coli cells grown under different redox conditions, it appeared that 89% of base substitutions were caused by the ROS, especially hydroxyl radicals, in cells growing in the atmosphere. The ROS-mediated spontaneous mutations included highly site-specific base substitutions, two types of randomly occurring transversions, G:C-->C:G and A:T-->T:A, and -1 frameshifts at non-iterated base sequences.     

The effect of camphorquinone (CQ) and CQ-related photosensitizers on the generation of reactive oxygen species and the production of oxidative DNA damage

Recent evidence suggests that following visible-light (VL) irradiation, CQ and the CQ-related photosensitizers benzil (BZ), benzophenone (BP), and 9-fluorenone (9-F) generate initiating radicals that may indiscriminately react with molecular oxygen forming reactive oxygen species (ROS). The purpose of this investigation was to determine whether VL-irradiated CQ, BZ, BP, and 9-F cause DNA damage due to the generation of ROS in vitro. ROS formation by CQ and CQ-related photosensitizers+/-dimethyl-p-toluidine (DMT) was investigated in a cell-free system with VL irradiation. DNA damage was determined using PhiX-174 RF I supercoiled double-stranded plasmid DNA and ROS quantified with 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO-9-AC), a fluorogenic ROS-sensitive probe. VL-irradiated CQ, BZ, BP, and 9-F (+/-DMT) produced significant DNA damage at 0.1, 0.5, and 1.0 mM and in a concentration-dependent manner (p<0.05). TEMPO-9-AC revealed that all investigated VL-irradiated photosensitizers produced significant amounts of ROS with BZ in the presence of DMT generating the most ROS after 30, 60, and 90 min. VL-irradiated CQ, BZ, BP, and 9-F +/-DMT continued to generate significant amounts of ROS 90 min after VL irradiation. As a result, future investigations should evaluate the effect of VL-irradiated photosensitizers in cells and possible protective effects provided by antioxidants.     

Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous studies, we have partly elucidated the mechanism of radiation-induced apoptosis of human peripheral T cells. The exact site of the ROS (reactive oxygen species) formation induced by irradiation has been so far unknown. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichlorodihydrofluorescein diacetate), DAPI, and Lysosensor. Our results showed that ROS formation apparently originated in the mitochondria and/or lysosomes instead of in the nuclei of irradiated T cells. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane instability, were observed at 1 h after 5 Gy irradiation of T cells. At 4 h after irradiation of 5 Gy, increase of fluorescence around the lysosomes, possibly revealing lysosomal rupture, was seen. Based on the above results, we concluded the possible existence of a new apoptotic cascade involving early lysosomal membrane destabilization in radiation-induced apoptosis of human peripheral T cells. Therefore, possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in radiation-induced T cell apoptosis.     

Polyamines reduce oxidative stress in Escherichia coli cells exposed to bactericidal antibiotics

Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.     

Synergistic DNA damage and lipid peroxidation in cultured human white blood cells exposed to 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and ultraviolet A

4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen in cigarette smoke, while ultraviolet (UV) irradiation from sunlight is a major factor for causing skin aging and skin cancer. However, little is known about the effects of the interaction between NNK and UV light on the induction of DNA damage and oxidative stress. In this study, we incubated human white blood cells (WBCs) with NNK, followed by irradiating the cells with ultraviolet A (UVA) (320-380 nm), and we measured DNA strand breaks (by the Comet or single-cell gel electrophoresis assay), lipid peroxidation (as thiobarbituric acid-reactive substances, TABRS), and the levels of intracellular reactive oxygen species (ROS). We found that preincubation with 1.0 mM NNK, followed by UVA irradiation (7.6 kJ/m2) synergistically increased DNA damage, lipid peroxidation, and the level of intracellular ROS in WBCs, while NNK or UVA alone had little or no effect. Electron spin resonance spectroscopic analyses showed that NNK plus UVA enhanced the UVA-induced generation of singlet oxygen but not hydroxyl radicals. In addition to ROS, bioactivation of NNK by cytochromes P450 (CYP) to form reactive NNK intermediates may also be involved in the synergistic damage to WBCs by NNK plus UVA. This is evidenced by the synergistic increase in N7-methylguanine (7-mGua), a major DNA adduct produced by NNK. Overall, the present study demonstrates that exposure of WBCs to both NNK and UVA synergistically increases DNA damage and lipid peroxidation and that such effects involve enhanced generation of ROS, especially singlet oxygen, and activation of NNK to 7-mGua by CYP. The results imply that NNK is a phototoxic agent.     

Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.     

槲皮素抑制异烟肼诱导的L-02细胞线粒体氧化应激性DNA损伤

目的:探讨活性氧(ROS)介导的线粒体氧化损伤在异烟肼(INH)诱导L-02细胞DNA损伤中的作用及槲皮素对细胞的保护作用。方法:建立体外培养INH致肝细胞L-02损伤的模型,将细胞分为对照(control)组、INH组、槲皮素低剂量(Que low)及高剂量(Que high)组。利用彗星试验评价细胞DNA损伤;制备L-02细胞线粒体,应用荧光探针DCFH-DA和rhodamine 123检测细胞线粒体ROS水平及线粒体膜电位(ΔΨm);采用TBA法测定丙二醛(MDA)含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)的活性;采用Western blotting法检测细胞中Bcl-2和Bax蛋白表达,计算Bax/Bcl-2值。结果:INH可诱导L-02细胞DNA损伤,使细胞线粒体ROS水平、细胞MDA含量及Bax/Bcl-2值明显增高,并使细胞ΔΨm值和SOD活性明显下降。而槲皮素能减轻细胞DNA损伤,减少细胞ROS水平,增加细胞ΔΨm值,降低细胞MDA含量,增加SOD活性,减少Bax/Bcl-2值。结论:INH可通过诱导细胞线粒体氧化应激导致L-02细胞DNA损伤。槲皮素能减轻INH诱导L-02细胞的DNA损伤,对L-02细胞具有保护作用,可能与其抑制ROS介导的线粒体氧化损伤有关。     

应用细胞损伤和反应指标评估体外氧化应激模型

目的应用细胞损伤及反应评估两种氧化应激模型在相关疾病研究中的应用。方法分别采用高糖和氧化剂Rosup培养HepG2肝细胞，检测细胞内活性氧量、DNA损伤及DNA修复酶PARP的表达。结果高糖培养4d可诱导细胞内活性氧生成增加至对照组3倍，Rosup组增加至对照组2倍。DNA损伤状况，高糖组是对照组2．7倍，Rosup组是对照组3倍。细胞反应指标PARP活性，高糖组是对照组2．8倍，Rosup组是对照组4．4倍。结论高糖诱导细胞的损伤表现为逐渐累积的过程，更适用于模拟体内长期复杂的损伤状态：而化学氧化剂是一个快速、强烈的诱导过程，更适用于研究急性氧化损伤过程。     

DNA damage induced by tumour necrosis factor-alpha in L929 cells is mediated by mitochondrial oxygen radical formation.

Treatment of L929 cells with tumour necrosis factor-alpha (TNF-alpha) plus actinomycin D induced DNA damage (indicated by the appearance of a sub-G1 peak due to extracellular leakage of low molecular weight DNA following DNA fragmentation) before significant cell lysis occurred. The DNA damage occurred in parallel with a decrease of the intracellular total glutathione content and an increase of intracellular reactive oxygen intermediates (ROI), as indicated by increased dihydrorhodamine 123 oxidation. Because the inhibition of mitochondrial respiration suppressed the increase of dihydrorhodamine 123 oxidation and DNA damage as well as the decrease in the total glutathione content, it was suggested that increased mitochondrial formation of ROI was responsible for DNA damage after TNF treatment. Deferoxamine (a ferric iron chelator) and dithiothreitol (a sulfhydryl reagent) both prevented DNA damage and cell killing, indicate that hydroxyl radicals generated from O2- and H2O2 produced by the mitochondria in a process catalysed by iron contributed to DNA damage and that this pathway may be involved in TNF-alpha-induced cytotoxicity. An inhibitor of poly(ADP)-ribose polymerase (3-aminobenzamide), worsened DNA damage, but was protective against cell lysis, suggesting that DNA repair subsequent to injury was more important than DNA damage per se in development of TNF-alpha cytotoxicity.     

Apoptosis induced by generated OH radicals inside cells after irradiation

OH radicals play a major role in radiation-induced DNA and cell membrane damage. These types of damage can also induce death by apoptosis through activation of a pro-apoptosis pathway. We attempted to detect OH radicals inside human promyelocytic leukemia (HL60) cells and estimate the relationship between radiation-induced apoptosis and OH radicals generated inside the cells. Electron spin resonance spectroscopy showed that OH radicals were generated by X-rays within irradiated cell pellets and the relative signal intensities of OH radicals increased with the radiation dose. Agarose gel electrophoresis revealed that the death of HL60 cells by apoptosis was accompanied by internucleosomal DNA fragmentation at 2 h after irradiation with 10-30 Gy. On ultrastructure evaluation by transmission electron microscopy, certain irradiated HL60 cells demonstrated condensed chromatin forms at the nuclear membrane and nuclear fragmentation. The frequency of apoptotic cells with condensation and fragmentation of nuclear chromatin increased with radiation dose in semithin sections. The increase of quantitative DNA fragmentation and percentage of non-living cells also correlated with radiation dose. These results suggest that OH radicals are generated inside cells before apoptosis occurs. The amount of OH radicals generated correlates with apoptotic cell death.     

Sources and mechanisms of cytoplasmic oxidative damage in Alzheimer's disease

While evidence supports a pathogenic and proximal role for oxidative stress in Alzheimer's disease, the causes and consequences of reactive oxygen species that promote oxidative damage have not been directly demonstrated. Co-incident with the reduced energy metabolism during the development of the disease, some of the key mitochondrial enzymes have shown deficient activity in AD neurons, which may lead to increased ROS production. However, we found that oxidative damage occurs primarily within the cytoplasm rather than in mitochondria. Given that SOD activity is increased in AD mitochondria and that metal ions such as iron and copper are enriched in susceptible neurons, we hypothesize that mitochondria, as a source, provide hydrogen peroxide, which, as an intermediate, once in the cytoplasm, will be converted into highly reactive hydroxyl radicals through Fenton reaction in the presence of metal ion and cause damage in cytoplasm.     

Age dependence of seizure-induced oxidative stress

The mechanisms underlying the decreased vulnerability of the immature brain to seizure-induced neuronal death remain unknown. We asked whether oxidative stress plays a role in the resistance of immature animals to seizure-induced brain damage. Mitochondrial aconitase inactivation and 8-hydroxy-2-deoxyguanosine (8-OHdG) were used as indices of steady-state mitochondrial superoxide (O(2)(-)) production and oxidative DNA damage, respectively. Kainate-induced seizures resulted in increased mitochondrial aconitase inactivation and 8-OHdG formation in adult (postnatal day 30 or more), but not in immature rats (postnatal days 12 and 21). Kainate administration did not induce manganese superoxide dismutase (MnSOD) or CuZnSOD in immature or adult rats. This developmental increase in mitochondrial O(2)(-) production and oxidative DNA damage following kainate seizures suggests that mitochondrial oxidative stress may be a key factor that renders the developing brain resistant to seizure-induced brain damage.