Saeko Tada-Oikawa

H2O2 Accelerates Cellular Senescence by Accumulation of Acetylated p53 via Decrease in the Function of SIRT1 by NAD+ Depletion

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD⁺-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H₂O₂ and examined DNA cleavage, depletion of intracellular NAD⁺, expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated β-galactosidase (SA-β-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H₂O₂, though no cell death was observed. NAD⁺ levels in TIG-3 cells treated with H₂O₂ were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD⁺ levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H₂O₂ treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H₂O₂ induced cell cycle arrest at 24h and increased SA-β-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Generation of hydrogen peroxide precedes loss of mitochondrial membrane potential during DNA alkylation-induced apoptosis

Pulsed field gel electrophoresis showed that the initiation time of DNA breakage induced by the DNA alkylating agent duocarmycin A, which is not a redox‐cycling agent, was almost the same in the human leukemia cell line HL‐60 and its H2O2‐resistant clone HP100. Catalase activity of HP100 cells was much higher than that of HL‐60 cells. Duocarmycin A‐mediated DNA ladder formation in HP100 cells was delayed compared with that in HL‐60 cells, suggesting the involvement of H2O2 in duocarmycin A‐induced apoptosis. Flow cytometry demonstrated that peroxide formation preceded loss of mitochondrial membrane potential (ΔΨm) in cells treated with duocarmycin A. Then, caspase‐3 was activated, followed by DNA ladder formation. These findings suggest that DNA damage by duocarmycin A induces H2O2 generation, which causes ΔΨm loss and subsequently caspase‐3 activation, resulting in apoptosis.

iNOS-dependent DNA damage via NF-κB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation‐mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535–42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV‐infected hamsters. We also examined the expression of nuclear factor‐κB (NF‐κB), which functions as a tumor promoter in inflammation‐associated cancer. Our results showed that although 1‐week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV‐induced 8‐nitroguanine and 8‐oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8‐oxodG level in the liver of OV‐infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF‐κB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV‐induced cholangiocarcinoma by inhibiting iNOS‐dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.

Proteomic identification of carbonylated proteins in the monkey hippocampus after ischemia–reperfusion

Reactive oxygen species (ROS) are known to participate in neurodegeneration after ischemia-reperfusion. With the aid of ROS, the calpain-induced lysosomal rupture provokes ischemic neuronal death in the cornu Ammonis (CA) 1 of the hippocampus; however, the target proteins of ROS still remain unknown. Here a proteomic analysis was done to identify and characterize ROS-induced carbonyl modification of proteins in the CA1 of the macaque monkey after transient whole-brain ischemia followed by reperfusion. We found that carbonyl modification of heat shock 70-kDa protein 1 (Hsp70-1), a major stress-inducible member of the Hsp70 family, was extensively increased before the neuronal death in the CA1 sector, and the carbonylation site was identified to be Arg469 of Hsp70-1. The CA1 neuronal death conceivably occurs by calpain-mediated cleavage of carbonylated Hsp70 that becomes prone to proteolysis with the resultant lysosomal rupture. In addition, the carbonyl levels of dihydropyrimidinase-like 2 isoform 2, glial fibrillary acidic protein, and beta-actin were remarkably increased in the postischemic CA1. Therefore, ischemia-reperfusion-induced oxidative damage to these proteins in the CA1 may lead to loss of the neuroprotective function, which contributes to neuronal death.

Endocrine disruptors that deplete glutathione levels in APC promote Th2 polarization in mice leading to the exacerbation of airway inflammation

Endocrine‐disrupting chemicals (EDC) are ubiquitous in environment and may have various undesirable effects on human health. In the present study, we have shown that some EDC [benzophenone, p‐octylphenol, and tributyltin chloride (TBT)] promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4+ T cells primed with anti‐CD3 and splenic antigen‐presenting cells (APC). The effect was indicated to be indirect via suppression of IL‐12 production and augmentation of IL‐10 production of APC, which are critical for the Th1 and Th2 development, respectively. Such modulation of cytokine production by EDC was associated with reduction of intracellular glutathione levels in APC. IL‐10 deprivation or the addition of N‐acetylcysteine, which replenishes intracellular glutathione level during priming, cancelled the effect of EDC on the promotion of Th2 polarization. Oral administration of TBT, which most effectively promoted Th2 polarization in vitro, exacerbated airway inflammation in a murine model of allergic asthma with concomitant enhancement of Th2‐type immunity. Collectively these results suggest that EDC such as benzophenone, p‐octylphenol, and TBT promote Th2 polarization indirectly via the depletion of glutathione in APC and subsequent modulation of IL‐10 and IL‐12 production that might result in the exacerbation of allergic diseases.

Epstein–Barr virus BHRF1 functions downstream of Bid cleavage and upstream of mitochondrial dysfunction to inhibit TRAIL-induced apoptosis in BJAB cells

We have previously reported that TNF-related apoptosis inducing ligand (TRAIL) causes cleavage of Bid via activation of caspase-8 and the loss of mitochondrial membrane potential (DeltaPsim), resulting in apoptosis. Experiments with BJAB clones expressing Epstein-Barr virus (EBV) anti-apoptotic protein BHRF1 showed that BHRF1 drastically inhibited TRAIL-mediated apoptosis. Although Western blot analysis demonstrated that TRAIL-induced Bid cleavage was not inhibited by BHRF1, the decrease in DeltaPsim caused by TRAIL was effectively blocked by BHRF1. These findings suggest that in BJAB cells, BHRF1 acts downstream of Bid cleavage and upstream of mitochondrial damage, resulting in inhibition of TRAIL-induced apoptosis.

Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation.

Metal oxide nanoparticles are widely used in industry, cosmetics, and biomedicine. However, the effects of exposure to these nanoparticles on the cardiovascular system remain unknown. The present study investigated the effects of nanosized TiO2 and ZnO particles on the migration and adhesion of monocytes, which are essential processes in atherosclerogenesis, using an in vitro set-up of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1). We also examined the effects of exposure to nanosized metal oxide particles on macrophage cholesterol uptake and foam cell formation. The 16-hour exposure to ZnO particles increased the level of monocyte chemotactic protein-1 (MCP-1) and induced the migration of THP-1 monocyte mediated by increased MCP-1. Exposure to ZnO particles also induced adhesion of THP-1 cells to HUVECs. Moreover, exposure to ZnO particles, but not TiO2 particles, upregulated the expression of membrane scavenger receptors of modified LDL and increased cholesterol uptake in THP-1 monocytes/macrophages. In the present study, we found that exposure to ZnO particles increased macrophage cholesterol uptake, which was mediated by an upregulation of membrane scavenger receptors of modified LDL. These results suggest that nanosized ZnO particles could potentially enhance atherosclerogenesis and accelerate foam cell formation.

[31] Determination of DNA damage, peroxide generation, mitochondrial membrane potential, and caspase-3 activity during ultraviolet A-induced apoptosis

Publisher Summary Solar ultraviolet (UV) light is a well-known carcinogen. UVB (280-320 nm) causes direct photo-activation of the DNA molecule to generate, mainly, pyrimidine photoproducts, leading to mutation and carcinogenesis. In addition to UVB, UVA (320-380 nm) has been implicated in multistage photocarcinogenesis. In UVA wavelengths, it is assumed that cellular DNA damage is produced indirectly through photosensitized reactions mediated by photosensitizers, as UVA can hardly be absorbed by the DNA. An excited endogenous photosensitizer molecule could react with DNA through direct electron transfer (type I) or through generation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion radical (type II). Thus, UVA, as well as UVB, may play an important role in the induction of carcinogenesis. Apoptosis contributes to the pathogenesis of a number of human diseases, including cancer. DNA damage can induce death by apoptosis that is characterized by cell shrinkage, chromatin condensation, and formation of oligonucleosome-length fragments of DNA (DNA ladder). UVA induces apoptosis in human and rodent cell lines. Roles for mitochondrial alterations and caspase activation in the regulation of apoptotic process have drawn much interest. A decrease in mitochondrial membrane potential (ΔΨm), causing opening of the permeability transition pore (PTP), has been implicated as a critical effector of apoptosis in a variety of cells. Subsequently, cytochrome-c released from mitochondria to cytosol initiates the activation of caspase-3. The activated caspase-3 can cleave an inhibitor of caspase-activated deoxyribonuclease (ICAD) and lead to DNA ladder formation. This chapter focuses on a methodological approach to study roles of DNA damage, peroxide generation, change of ΔΨm, and caspase-3 activity in a UVA-induced apoptotic pathway.

Identification of a Glutamic Acid Repeat Polymorphism of ALMS1 as a Novel Genetic Risk Marker for Early-Onset Myocardial Infarction by Genome-Wide Linkage Analysis

Background—Myocardial infarction (MI) is a leading cause of death worldwide. Given that a family history is an independent risk factor for coronary artery disease, genetic variants are thought to contribute directly to the development of this condition. The identification of susceptibility genes for coronary artery disease or MI may thus help to identify high-risk individuals and offer the opportunity for disease prevention. Methods and Results—We designed a 5-step protocol, consisting of a genome-wide linkage study followed by association analysis, to identify novel genetic variants that confer susceptibility to coronary artery disease or MI. A genome-wide affected sib-pair linkage study with 221 Japanese families with coronary artery disease yielded a statistically significant logarithm of the odds score of 3.44 for chromosome 2p13 and MI. Further association analysis implicated Alström syndrome 1 gene (ALMS1) as a candidate gene within the linkage region. Validation association analysis revealed that representative single-nucleotide polymorphisms of the ALMS1 promoter region were significantly associated with early-onset MI in both Japanese and Korean populations. Moreover, direct sequencing of the ALMS1 coding region identified a glutamic acid repeat polymorphism in exon 1, which was subsequently found to be associated with early-onset MI. Conclusions—The glutamic acid repeat polymorphism of ALMS1 identified in the present study may provide insight into the pathogenesis of early-onset MI.

Proteomic analysis of carbonylated proteins in the monkey substantia nigra after ischemia-reperfusion

Abstract In Parkinsons disease (PD), oxidative stresses cause cell death of dopaminergic neurons of the substantia nigra (SN), but its molecular mechanism still remains unclarified. Our previous study of proteomic analysis in the monkey CA1 hippocampus after ischemia–reperfusion revealed reactive oxygen species (ROS)-induced carbonyl modification of a molecular chaperone, heat shock 70-kDa protein 1 (Hsp70.1), especially in its key site, Arg469. Here, to clarify the mechanism of neurodegeneration in PD, the SN tissue of the same monkey experimental paradigm was studied for identifying and characterizing carbonylated proteins by the two-dimensional gel electrophoresis with immunochemical detection of protein carbonyls (2D Oxyblot). We found carbonyl modification not only of Hsp70.1 but also of mitochondrial aconitase, dihydropyrimidinase-related protein 2, T-complex protein 1 subunit alpha, dihydrolipoyl dehydrogenase, fructose-bisphosphate aldolase C, glutamate dehydrogenase 1, and aspartate aminotransferase. Intriguingly, in the SN also, the carbonylation site of Hsp70.1 was identified to be Arg469. Since Hsp70.1 is recently known to stabilize the lysosomal membrane, its oxidative injury conceivably plays an important role in the ROS-mediated neuronal cell death by inducing lysosomal destabilization. Implications of each carbonylated proteins for the dopaminergic neuronal death were discussed, in comparison with the CA1 neuronal death.