Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5'-GG-3', 5-GGGG-3', 5'-GGGGG-3' sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H₂O₂ and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.     

Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.     

Protective effect of phytic acid on oxidative DNA damage with reference to cancer chemoprevention.

Phytic acid (myo-inositol hexaphosphate) is one of the most promising cancer chemopreventive agents. We investigated the mechanism by which phytic acid expresses preventive action to cancer. Phytic acid inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cultured cells treated with an H2O2-generating system, although it did not scavenge H2O2. Site-specific DNA damage by H2O2 and Cu(II) at GG and GGG sequences was inhibited by phytic acid, but not by myo-inositol. Phytic acid alone did not cause DNA damage and thus, it should not act as a prooxidant. We conclude that phytic acid acts as an antioxidant to inhibit the generation of reactive oxygen species from H2O2 by chelating metals, resulting in chemoprevention of cancer.     

Cellular factor stimulating DNA synthesis in nuclei isolated from sea urchin embryos

DNA synthesis in isolated nuclei of morula-stage embryos of sea urchin was studied. Embryonic extracts of cleaving embryos (but not unfertilized eggs) stimulated DNA synthesis in the in vitro system. A stimulatory factor was identified which eluted at 0.52 M KCl during chromatography on DEAE-cellulose column. This factor was inactivated by heat treatment and trypsin digestion, and was resolved into three active peaks by gel filtration (Stokes radii of 6.3, 4.6, and 4.1 nm, respectively).     

DNA repair in Bloom's syndrome fibroblasts after UV irradiation or treatment with mitomycin C

Sensitivities to UV and mitomycin C (MC) of fibroblasts obtained from 3 Japanese patients with Bloom's syndrome (BS) were studied. One BS strain was more sensitive to UV than normal cells only in colony-forming ability. Other responses to UV, such as unscheduled DNA synthesis, host-cell reactivation and removal of UV-endonuclease-susceptible sites, were normal in all 3 strains. These BS strains were more sensitive to MC than were normal cells. However, the amounts of unscheduled DNA synthesis after treatment with MC in BS cells did not differ from those in normal cells.     

Histochemical Detection of Arylsulfatase Activity in Sea Urchin Embryos

Localization of arylsulfatase activity in the sea urchin embryo was determined histochemically by light and electron microscopy. Histochemical observations by light microscopy revealed that the arylsulfatase activity appears after the gastrula stage and that it is restricted to the cells of the aboral ectoderm. The enzyme activity is mainly located in the apical cellular cytoplasm and is associated with lysosome-like structures that are frequently fused with yolk granules. Intense activity is also detected in the region of the endoplasmic reticulum and Golgi apparatus. No enzyme activity is found in the extracellular spaces of embryos.     

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4beta-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H2O2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H2O2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H2O2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) demonstrated that procyanidin B2 decreased the signal of M4PO-OH from H2O2 and Fe(II), whereas procyanidin B2 enhanced the signal from H2O2 and Cu(II). As an antioxidant mechanism, UV-visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H2O2 and metal ions.     

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.     

Kinematic analysis of bipedal locomotion of a Japanese macaque that lost its forearms due to congenital malformation

Spontaneously acquired bipedal locomotion of an untrained Japanese monkey (Macaca fuscata) is measured and compared with the elaborated bipedal locomotion of highly trained monkeys to assess the natural ability of a quadrupedal primate to walk bipedally. The subject acquired bipedalism by himself because of the loss of his forearms and hands due to congenital malformation. Two other subjects are performing monkeys that have been extensively trained for bipedal posture and locomotion. We videotaped their bipedal locomotion with two cameras in a lateral view and calculated joint angles (hip, knee, and ankle) and inertial angle of the trunk from the digitized joint positions. The results show that all joints are relatively more flexed in the untrained monkey. Moreover, it is noted that the ankle is less plantar flexed and the knee is more flexed in mid-to-late stance phase in the untrained monkey, suggesting that the trunk is not lifted up to store potential energy. In the trained monkeys, the joints are extended to bring the trunk as high as possible in the stance phase, and then stored potential energy is exchanged for kinetic energy to move forward. The efficient inverted pendulum mechanism seems to be absent in the untrained monkeys locomotion, implying that acquisition of such efficient bipedal locomotion is not a spontaneous ability for a Japanese monkey. Rather, it is probably a special skill that can only be acquired through artificial training for an inherently quadrupedal primate.This revised version was published online in April 2005 with corrections to the cover date of the issue.