Kimiko Izumo

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.

In vivo expression of proinflammatory cytokines in HIV encephalitis: an analysis of 11 autopsy cases

As the pathogenesis of AIDS dementia complex (ADC), cytokines such as TNF‐α and IL‐1β have been thought to have toxic effects on CNS cells and induce neuronal cell death. However, many of the discussions have been based on the studies done by in vitro experiments. There are only a few reports which demonstrate proinflammatory cytokines directly in vivo in HIV encephalitis (HIVE) brains, and roles of these cytokines with relation to HIV‐1 infection are not yet clarified. In the present study, we examined 11 autopsy cases of HIVE using immunohistochemistry, and explored which cell types expressed these cytokines and whether expression of cytokines was related to viral infection. IL‐1β was detected in the frontal white matter of all 11 cases where microglial nodules were observed to varying degrees, whereas TNF‐α was detected in seven cases. IL‐1β− or TNF‐α‐positive cells were almost restricted to CD68‐positive macrophages/microglia and mild expression of these cytokines by astrocytes was observed in two cases with severe HIVE. IL‐1β was detected in some HIVp24‐positive multinucleated giant cells. However, we could not detect TNF‐α expression in the HIVp24‐positive cells, which indicates that IL‐1β is induced by HIV‐1 infection. In conclusion, a macrophage/microglia lineage is the main cell type to release cytokines in HIVE, and IL‐1β expression by HIV‐1‐infected cells may be one of the important factors for induction of HIVE. In addition, many non‐infected macrophages/microglia as well as some astrocytes express IL‐1β and TNF‐α, which might contribute to pathogenesis of ADC.

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H2O2, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.

Benzene induces cytotoxicity without metabolic activation.

Benzene Induces Cytotoxicity without Metabolic Activation: Takuro Nishikawa, et al. Departments of Environmental Medicine and Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University—

Benzene Metabolite 1,2,4-Benzenetriol Induces Halogenated DNA and Tyrosines Representing Halogenative Stress in the HL-60 Human Myeloid Cell Line

Background: Although benzene is known to be myelotoxic and to cause myeloid leukemia in humans, the mechanism has not been elucidated. Objectives: We focused on 1,2,4-benzenetriol (BT), a benzene metabolite that generates reactive oxygen species (ROS) by autoxidation, to investigate the toxicity of benzene leading to leukemogenesis. Methods: After exposing HL-60 human myeloid cells to BT, we investigated the cellular effects, including apoptosis, ROS generation, DNA damage, and protein damage. We also investigated how the cellular effects of BT were modified by hydrogen peroxide (H2O2) scavenger catalase, hypochlorous acid (HOCl) scavenger methionine, and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase (MPO)-specific inhibitor. Results: BT increased the levels of apoptosis and ROS, including superoxide (O2•−), H2O2, HOCl, and the hydroxyl radical (•OH). Catalase, ABAH, and methionine each inhibited the increased apoptosis caused by BT, and catalase and ABAH inhibited increases in HOCl and •OH. Although BT exposure increased halogenated DNA, this increase was inhibited by catalase, methionine, and ABAH. BT exposure also increased the amount of halogenated tyrosines; however, it did not increase 8-oxo-deoxyguanosine. Conclusions: We suggest that BT increases H2O2 intracellularly; this H2O2 is metabolized to HOCl by MPO, and this HOCl results in possibly cytotoxic binding of chlorine to DNA. Because myeloid cells copiously express MPO and because halogenated DNA may induce both genetic and epigenetic changes that contribute to carcinogenesis, halogenative stress may account for benzene-induced bone marrow disorders and myeloid leukemia.

Impaired Astrocytes and Diffuse Activation of Microglia in the Cerebral Cortex in Simian Immunodeficiency Virus-Infected Macaques Without Simian Immunodeficiency Virus Encephalitis

Various types of neuronal damage have been reported in acquired immunodeficiency syndrome (AIDS) dementia. We previously demonstrated that inflammation and cortical damage occur independently according to viral tropism in a simian immunodeficiency virus (SIV)-infected macaque model of AIDS dementia. To elucidate the pathogenesis of cortical degeneration, we examined the frontal cortex of SIV-infected macaques and found apoptosis and decreased expression of the excitatory amino acid transporter 2 in astrocytes and diffuse activation of microglia in association with limited neuronal damage. Some activated microglia also expressed excitatory amino acid transporter 2 but not proinflammatory cytokines. No inflammatory changes were seen in the cortex or the white matter, and SIV-infected cells were not detected in or around cortical lesions either by immunohistochemistry or by the polymerase chain reaction detection of SIV genomes of extracted DNA from microdissected tissue samples. These results indicate that an astrocytic abnormality and a compensatory activation of microglia might provide a protective effect against neuronal degeneration in the frontal cortex of SIV-infected macaques without SIV encephalitis.

Decrease of aquaporin-4 and excitatory amino acid transporter-2 indicate astrocyte dysfunction for pathogenesis of cortical degeneration in HIV-associated neurocognitive disorders.

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV‐associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter‐2 (EAAT‐2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian‐human immunodeficiency virus (SHIV)‐infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543‐3‐infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV‐infected macaques, three SIVsm543‐3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte‐specific protein aquaporin‐4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV‐infected macaques, which was associated with EAAT‐2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT‐2. Some astrocytes express EAAT‐2 but not AQP4, and decrease of EAAT‐2 expression tended to be less than the decrease of AQP4. Active‐caspase‐3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT‐2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT‐2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT‐2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.

Snail modulates cell metabolism in MDCK cells

Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of key enzymes. This results in enhanced glucose dependency and leads to cell death under low-glucose conditions. On the other hand, the reduced requirements for oxygen and nutrients from the surrounding environment, might confer the resistance to cell death induced by hypoxia and malnutrition.

Characterization of glycans of CD4+T cells in HAM/TSP

The outermost structure of cells is galectin-glycan lattice, that is shown to have many roles in cell-cell interactions. Recently, biofilm-like extracellular viral assemblies were shown to mediate HTLV-1 cell-to-cell transmission. Here, we tried to characterize the glycans on the CD4+T cells in HAM. CD4+T cells from four respective cases of HAM, AC(asymptomatic carriers), and NC(negative control) were subjected to lectin array analysis. Similarly, glycans liberated from membrane proteins of CD4+T cells from six respective cases of HAM and NC analyzed by MALDI-TOF MS. We found that UDA(Urtica dioica agglutinin) and STL(Solanum tuberosum lectin (Potato)) lectins, that recognize N-glycan polylactosamine which consist of repeats of the disaccharide betaGal(1-4)betaGlcNAc(1-3), were significantly high in HAM in lectin array analysis. Interestingly, UDA was reported to inhibit cell-to-cell infection in vitro. On the other hand, MALDI-TOF MS analysis found several O-glycans in HAM. Candidates verified in GlycoSuite database were mainly O-glycans attach to MUC1 and Leukosialin(CD43) as carrier proteins. These proteins were reported to play roles in ATL and cell-to-cell infection as well. These glycans and carrier proteins may be therapeutic target of HAM.