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.     

Semiaquatic Heteroptera locomotion: coral treaders (Hermatobates weddi, Hermatobatidae), sea skaters (Halovelia septentrionalis, Veliidae), and water striders (Metrocoris histrio, Gerridae). Usual and unusual gaits

Using high-speed video recordings, we carried out an analysis of the locomotion gaits of the following aquatic Heteroptera: coral treaders Hermatobates weddi (Hermatobatidae), sea striders Halovelia septentrionalis (Veliidae), and water striders Metrocoris histrio (Gerridae), in the Island of Amami Oshima, Kagoshima Prefecture, Japan. Most insects use an alternating double tripod gait for walking, whereas species of Gerridae and some Veliidae use a synchronous rowing gait. We found that H. weddi used a peculiar locomotion gait, a modification of the double tripod gait. In this special gait, two alternating dipods (mid and hind legs) are used, while the forelegs remained inactive. Contralateral mid and hind stroked simultaneously. The mid leg recovered immediately after the stroke; however, the hind leg was delayed and remained extended after the stroke. Next, the following bipod stroked, and when that mid leg finished the stroke, both ipsilateral mid and hind (the one which did not recover after the stroke) legs recovered together. Turning is also unique in H. weddi because the body axis rotation and the course turning (deflection) were clearly separated in two phases. We compared the kinematics of H. weddi pattern with the synchronous rowing pattern found in H. septentrionalis and M. histrio and discussed some biomechanical consequences. We also analyzed phylogenetic implications of this gait, and we posit that the modified double dipod gait is a uniquely derived character of the family Hermatobatidae. The synchronous rowing gait would be an autapomorphy for the clade Gerridae + Veliidae. The modified thorax, with the meso and metacoxae horizontally directed, would be a synapomorphy for the superfamily Gerroidea (Hermatobatidae, Gerridae, and Veliidae). Handling editor: Koen Martens     

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.     

Deduced primary structure of rat tryptophan-2,3-dioxygenase

The complete amino acid sequence of the tryptophan 2,3-dioxygenase (TO) of rat liver was determined from the nucleotide sequence of a full length TO cDNA isolated from a rat liver cDNA library and determined its primary structure. TO was encoded in a mRNA of about 1.7 kb containing an open reading frame of 1218 bp. According to the deduced amino acid sequence, the monomeric polypeptide of TO consisted of 406 amino acid residues with a calculated molecular weight of 47,796 daltons. It has twelve histidine residues around its hydrophobic region, which has homology with some heme proteins and oxygenase, suggesting that this hydrophobic region might to be the core of TO for the activity.     

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.