Rensuke Goto

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.

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.

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.

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.

Effects of alkyl glycosides incorporated into liposomes prepared from synthetic amphiphiles on their tissue distribution in Ehrlich solid tumor-bearing mice

A study of the effects of alkyl glycosides incorporated into synthetic liposomes with respect to their stability, their in vivo distribution in Ehrlich solid tumor-bearing mice and their in vitro interaction with liver cells was undertaken. The synthetic liposomes were prepared from N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C12) and labeled with 99mTc. n-Dodecyl glucoside (DG) and n-dodecyl sucrose (DS) were used as alkyl glycosides. The stability was hardly changed by incorporation of alkyl glycosides into the liposomes in saline and serum. The uptake of DG- and DS-modified N+C5Ala2C12 liposomes decreased in liver and spleen compared with that of unmodified N+C5Ala2C12 liposomes, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, where the DS-modified N+C5Ala2C12 liposomes had a marked tendency. It was observed with electron micrographs that the size of N+C5Ala2C12 liposomes became small by incorporation of alkyl glycoside. The smaller N+C5Ala2C12 liposomes were found to result in the lower uptake in liver. The interaction of the liposomes with liver cells in vitro indicated that both DG- and DS-modified liposomes had a low affinity for liver cells compared with the unmodified liposomes and the extent of interaction of the DS-modified liposomes was weaker than that of the DG-modified liposomes.

Phosphatidylserine induces apoptosis in CHO cells without mitochondrial dysfunction in a manner dependent on caspases other than caspases-1, -3, -8 and -9.

Treatment of Chinese hamster ovary K1 cells with phosphatidylserine (PS) caused typical apoptosis with distinct morphological and biochemical features in a dose‐ and time‐dependent manner. However, unlike camptothecin‐induced apoptosis, changes in mitochondrial transmembrane potential were not observed. In addition, cytochrome c release did not occur in PS‐induced apoptosis. A pan caspase inhibitor, Z‐VAD, significantly inhibited the apoptosis, but inhibitors of caspase‐1, ‐3, ‐8 and ‐9 did not. Activities of caspase‐1, ‐3, ‐8 and ‐9 were increased by treatment of the cells with camptothecin, but not with PS. These results suggest that PS‐induced apoptosis occurs without the collapse of mitochondrial transmembrane potential and without the release of cytochrome c, in a manner independent of caspase‐1, ‐3, ‐8 and ‐9.

Chemical properties of technetium-99m-DL-homocysteine, a possible tumor-imaging agent

The chemical properties of99mTc-DL-homocysteine (99mTc-Hcy) showing high accumulation in several experimental tumors were investigated. The form of tumor-tropic99mTc-Hcy was a polymeric complex which appeared at void volume on Sephadex G-15 by eluting with 5 mM Hcy. This complex changed into smaller complexes of ca. 600 molecular weight in the presence of 150 mM NaCl and 5 mM Hcy, suggesting that99mTc-Hcy was a complex composed of smaller polymers which are weakly bound together by an ionic bond. The complex showed a negative charge. The Hcy/Tc molar ratio in the complex was approximately 2 and no Sn was detected.

IUPAC-NIST Solubility Data Series. 90. Hydroxybenzoic Acid Derivatives in Binary, Ternary, and Multicomponent Systems. Part I. Hydroxybenzoic Acids, Hydroxybenzoates, and Hydroxybenzoic Acid Salts in Water and Aqueous Systems

The solubility data for well-defined binary, ternary, and multicomponent systems of solid-liquid type are reviewed. One component, which is 2-, 3-, and 4-hydroxybenzoic acids, 4-hydroxybenzoate alkyl esters (parabens), or hydroxybenzoic acid salts, is in the solid state at room temperature and another component is liquid water, meaning that all of the systems are aqueous solutions. The ternary or multicomponent systems include organic substances of various classes (hydrocarbons of several structural types, halogenated hydrocarbons, alcohols, acids, ethers, esters, amides, and surfactants) or inorganic substances. Systems reported in the primary literature from 1898 through 2000 are compiled. For seven systems, sufficient binary data for hydroxybenzoic acids or parabens in water are available to allow critical evaluation. Almost all data are expressed as mass and mole fractions as well as the originally reported units, while some data are expressed as molar concentration.

The Antiapoptotic Effect of Low-dose UVB Irradiation in NIH3T3 Cells Involves Caspase Inhibition¶

UVB irradiation is a well‐known apoptosis induction factor. However, we have previously found that low doses of UVB irradiation inhibited apoptosis induced by both serum starvation and lack of extracellular matrix, involving a significant inhibition of caspase‐3/7 activation. In this study, we report on the relationship between the UVB‐induced antiapoptotic effect and caspase‐3/7 inhibition by reactive oxygen species (ROS). The UVB‐induced antiapoptotic effect was partially prevented by an antioxidant agent, N‐acetylcysteine. A ROS‐generating agent, menadione and a pro‐oxidant agent, H2O2 also showed an effect that was similar to the UVB‐induced antiapoptotic effect, indicating that ROS contributed to the antiapoptotic effect. UVB irradiation significantly suppressed caspase‐3/7 activation, which was caused by the inhibition of proteolysis and not by the inhibition of enzymatic activity itself. The prevention of proteolysis was also confirmed by both the following results: one is the inhibition of in vitro caspase‐3/7 and −9 activation in cell lysates exposed to UVB in the presence of cytochrome c and dATP, which was caused by the production of ROS, and the other is the inhibition of in vitro caspase‐3/7 activation in the presence of active caspase‐9. These results showed that the inhibition of the caspase cascade downstream mitochondria by ROS production, leading to a significant inhibition of caspase‐3/7 activation, was one of the causes of the antiapoptotic effect by small doses of UVB irradiation.