Yuko Ibuki

Enhancement of NO Production From Resident Peritoneal Macrophages by In Vitro γγ-Irradiation and Its Relationship to Reactive Oxygen Intermediates

The functional changes in macrophages (Mphi) following exposure to a high dose (6 Gy) of gamma-rays in vitro were investigated. Resident peritoneal Mphi obtained from C57BL/6 mice were irradiated with gamma-rays (137Cs, 0.3 Gy/min). High-dose irradiation enhanced nitric oxide (NO) production from Mphi treated with interferon-gamma and their cytotoxic activity. The enhancement of NO production by irradiation was attributed to high levels of expression of the inducible nitric oxide synthase. Furthermore, the participation of reactive oxygen intermediates in NO production was examined. Nitric oxide production was not enhanced by treatment with the membrane-oxidizing agent tert-butyl hydroperoxide or the hypoxanthine/xanthine oxidase superoxide (O2.-)-generating system. On the other hand, NO production was enhanced by treatment with a low dose of hydrogen peroxide (H2O2), which can diffuse passively through the cell membrane and can be converted into hydroxyl radicals (HO.) that cause DNA breaks. In addition, treatment with low-dose actinomycin D, which induces DNA strand breaks, enhanced NO production, but hydroxyurea, which stops DNA replication without DNA strand breaks, had no such effect. These findings suggest that DNA strand breaks caused by hydroxyl radicals formed inside the cells by gamma-irradiation, or strand breaks caused directly by radiation, plays an important role in the enhancement of NO production, but peroxidation of cell membranes has little effect.

DNA damage induced by coexposure to PAHs and light.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment as pollutants in air, water and soil, and some are carcinogenic, being associated with various types of cancer. A majority of the research concerning the biological effects of PAHs has focused on the metabolic activation and DNA adducts leading to mutation and transformation. Although the role of the PAHs as photosensitizers has received much less attention, investigators have shown that PAHs excited by sunlight induced significant cytotoxicity and several kinds of DNA damage. Some PAHs were recently proved to be photomutagenic. In this review, we discuss the influence of PAHs in combination with sunlight focusing on the phototoxicity and cellular DNA damage produced.

Beneficial effects of curcumin on antitumor activity and adverse reactions of doxorubicin

Many medicines used in cancer chemotherapy decrease the quality of life (QOL). It is believed that an increase in food intake during cancer chemotherapy may produce an improvement in QOL. Curcumin is widely used as a coloring and flavoring agent in food. The effects of curcumin in relation to the chemotherapeutic drug doxorubicin (DOX) were examined. While DOX alone did not decrease tumor weight, the combination of DOX and curcumin significantly reduced tumor weight to 56.5% (p<0.05) of that of the control group. The combined curcumin enhanced apoptosis by DOX and decreased cell viability. The curcumin-DOX combination also suppressed activation of caspase-3, -8, and -9 compared to DOX alone. It is presumed that combining curcumin increased DOX-induced antitumor activity by suppressing the main caspase pathway and activating the main caspase independent pathway. The combination of curcumin and DOX suppressed the reduction of glutathione peroxidase activity and increased lipid peroxide levels in the heart. Therefore, it is expected that curcumin may reduce the adverse reactions associated with DOX. Our results suggest that curcumin can be used as a modulator to enhance the therapeutic index of cancer patients and improve their QOL.

γ-Irradiation-induced DNA damage enhances NO production via NF-κB activation in RAW264.7 cells

Abstract We investigated the mechanism of augmentation of nitric oxide (NO) production in the murine macrophage cell line RAW264.7 after γ-irradiation. The cells treated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS) showed enhanced NO production by γ-irradiation in a dose-dependent manner, accompanying the induction of inducible nitric oxide synthase (iNOS) expression. Nuclear factor kappa B (NF-κB) activation was induced 1 h after γ-irradiation dose-dependently, which was detected by the degradation of I-κB. Inhibitors of I-κB degradation, MG132 and N α - p -tosyl- l -lysine chloromethyl ketone (TLCK), suppressed the further increase by γ-irradiation in IFN-γ-induced NO production, showing that γ-irradiation induced NO production via NF-κB activation. Although NF-κB is known to be a redox-sensitive transcription factor, the antioxidant agents N -acetyl-cysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox) showed no suppression and treatment with H 2 O 2 showed only slight enhancement of IFN-γ-induced NO production. The DNA damaging agents camptothecin and etoposide enhanced IFN-γ-induced NO production and showed I-κB degradation, indicating that the increase in NO production was due to direct DNA damage. Furthermore, 3-aminobenzamide (3AB) and benzamide, inhibitors of poly (ADP-ribose) polymerase (PARP) that are activated upon recognition of DNA strand breaks, suppressed the further increase by γ-irradiation in IFN-γ-induced NO production and the I-κB degradation by γ-irradiation. We concluded that (1) the increase in NO production was due to direct DNA damage by γ-irradiation, and that (2) PARP activation through DNA damage induced NF-κB activation, leading to iNOS expression and NO production.

Coexposure to benzo(a)pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed γ‐H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that coexposure to benzo[a]pyrene (BaP), a wide‐spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether coexposure to BaP and UVA generates γ‐H2AX in CHO‐K1 cells. Single treatment with BaP (10−9–10−7 M) or UVA (∼2.4 J/cm2) did not result in γ‐H2AX, however, coexposure drastically induced foci of γ‐H2AX in a dose‐dependent manner. γ‐H2AX could be detected even at very low concentration of BaP (10−9 M) plus UVA (0.6 J/cm2), which did not change cell survival rates. NaN3 effectively inhibited the formation of γ‐H2AX induced by coexposure, indicating the contribution of singlet oxygen. This is the first evidence that coexposure to BaP and UVA induced DSBs, involving γ‐H2AX.

Antiapoptotic Effects Induced by Different Wavelengths of Ultraviolet Light

Cells receive signals for survival as well as for death, and the balance between the two ultimately determines the fate of the cells. UV‐triggered apoptotic signaling has been well documented, whereas UV‐induced survival effects have received little attention. We have reported previously that UVB irradiation prevented apoptosis, which is partly dependent on activation of the phosphatidylinositol 3‐kinase (PI3‐kinase)–Akt pathway (Ibuki Y. and Goto, R. [2000] Biochem. Biophys. Res. Commun. 279, 872–878). In this study, antiapoptotic effects and survival signals of UV with different wavelength ranges, UVA, UVB and UVC, were examined. NIH3T3 cells showed apoptotic cell death by detachment from the extracellular matrix under serum‐free conditions, which was prevented by all wavelengths. However, the effect of UVA was less than those of UVB and UVC, as determined by metabolism of fluoresceine diacetate and the appearance of chromatin‐condensed cells. Furthermore, the effects of three wavelengths of UV on the apoptotic pathway upstream of the nuclear signals were examined. Reduction of mitochondrial transmembrane potential (ΔΨ) and activation of caspase‐9 and ‐3 were suppressed by all three wavelengths of UV, showing wavelength‐dependent effects as mentioned previously. Shorter wavelengths showed stronger inhibitory effects on caspase‐8 activity. The PI3‐kinase inhibitor wortmannin partially inhibited the UVB‐ and UVC‐induced suppression of apoptosis but not the inhibitory effect of UVA. Furthermore, normal ΔΨ maintained by UVA was not changed in the presence of wortmannin, but those by UVB and UVC were reduced. Akt was clearly phosphorylated by all three wavelengths. The phosphorylation by UVB and UVC was completely inhibited by addition of wortmannin, but that by UVA was not, in agreement with the results of survival and of ΔΨ. These results suggested the existence of two different survival pathways leading to suppression of apoptosis, one for UVA that is independent of the PI3‐kinase–Akt pathway and the other for UVB and UVC that is dependent on this pathway.

Phototoxicity of benzo[a]pyrene by ultraviolet A irradiation: induction of apoptosis in Jurkat cells.

The toxicology of benzo[a]pyrene (BaP) has been mainly studied with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of BaP, there have been no reports regarding the pattern of cell death induced by this agent. In this study, we investigated the pattern and mechanism of cell death induced by coexposure to BaP plus ultraviolet A (UVA) in Jurkat cells. Coexposure to BaP plus UVA showed dose-dependent cytotoxicity. The pattern of cell death was apoptotic as determined by cell shrinkage, chromatin condensation, appearance of subdiploid apoptotic nuclei and translocation of phosphatidylserine to the outer membrane leaflet. Coexposure also strongly increased caspase-3/7 activity and slightly elevated those of caspase-8/6 and -9. The pan caspase inhibitor Z-VAD-CH(2)-DCB partially inhibited the phototoxicity of BaP. Cytochrome c release was observed 6 h after coexposure, but not after 1 h. Furthermore, the phototoxicity was inhibited by NaN(3) (quencher of singlet oxygen), but not by mannitol (quencher of hydroxy radicals). Chromatin condensation and translocation of phosphatidylserine were also inhibited by NaN(3), suggesting that the induction of apoptosis by coexposure to BaP plus UVA was due to singlet oxygen production.

Preparation of DNA-adsorbed TiO2 particles--augmentation of performance for environmental purification by increasing DNA adsorption by external pH regulation.

We have previously developed a novel photocatalyst, DNA-attached titanium dioxide (DNA-TiO(2)), useful for the recovery and decomposition of chemicals [Suzuki et al. Environ. Sci. Technol. 42, 8076, 2008]. Chemicals accumulated in DNA near the surface of TiO(2) and were degraded under UV light. The efficiency of their removal was dependent on the amount of DNA adsorbed on TiO(2), indicating the attachment of larger amounts of DNA to result in higher efficiency. In this study, we succeeded in improving the performance of DNA-TiO(2) by increasing the amount of DNA adsorbed by regulating the external pH. The adsorption of DNA by TiO(2) dramatically increased at pH2, to about fourfold that at other pH values (pH4-10). Repeating the process of DNA addition increased the adsorption further. The attached DNA was stable on the surface of TiO(2) at pH2-10 and 4-56 degrees C, the same as DNA-TiO(2) prepared at pH7. As the DNA-TiO(2) prepared at pH2 retained much DNA on its surface, chemicals (methylene blue, ethidium bromide, etc.) which could intercalate or react with DNA were effectively removed from solutions. The photocatalytic degradation was slow at first, but the final degradation rate was higher than for non-adsorbed TiO(2) and DNA-TiO(2) prepared at pH7. These results indicated that preparation of DNA-TiO(2) at pH2 has advantages in that much DNA can be attached and large amounts of chemicals can be concentrated in the DNA, resulting in extensive decomposition under UV light.

Immunomodulatory effects of ultraviolet B irradiation on atopic dermatitis in NC/Nga mice.

Background: Atopic dermatitis (AD) is a common pruritic inflammatory skin disease, which occurs primarily in childhood. Recently, narrow‐band ultraviolet B (UVB) phototherapy has been used to treat AD, but the mechanism involved is unknown. In this study, we investigated whether UVB irradiation influences AD in the NC/Nga mouse.

Proteome Analysis of UV‐B‐Induced Anti‐apoptotic Regulatory Factors¶

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low‐dose UV‐B irradiation (∼0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low‐dose UV‐B‐irradiated NIH3T3 cells, using two‐dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI‐TOF/TOF‐MS, some as structural proteins. Although typical apoptosis‐related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.