Motohisa Kaneko

Cigarette smoke induces DNA single-strand breaks in human cells

Epidemiological evidence suggests that smoking is a major cause of human lung cancer1,2. However, the mechanism by which cigarette smoke induces the cancer remains obscure, although in tobacco Carcinogenesis, promotion and/or co-carcinogenesis may have crucial roles. The epidemiological data show that if an individual stops smoking, the risk of his contracting lung cancer increases no further. Moreover, laboratory experiments show that cigarette smoke condensate (CSC) exhibits co-carcinogenic and promoting activities in tumour production and malignant transformation3–5. Clastogenic action is thought to be intimately involved in tumour promotion6–8, and it is therefore interesting that visible chromosome changes such as chromosome aberrations and sister chromatid exchanges are known to be caused by cigarette smoke9–11. However, there has been no previous direct demonstration that cigarette smoke can cause single-strand breaks (SSB) in DNA. Here we report that cigarette smoke induces considerable numbers of DNA SSB in cultured human cells, and that such strand breaks may be ascribed to active oxygen generated from cigarette smoke.

Effects of diethylstilbestrol and its methyl ethers on aneuploidy induction and microtubule distribution in Chinese hamster V79 cells

We previously reported that diethylstibestrol (DES) and its derivatives inhibit the in vitro polymerization of microtubule proteins isolated from porcine brain (Sato et al., 1987). We found that the presence of the free hydroxy group of DES was indispensable for the inhibition of microtubule assembly. In the present investigation, this structure-activity relationship was confirmed by the effects of DES and its methyl ethers on chromosome number and the cellular microtubule architecture of Chinese hamster V79 cells, revealed by fluorescent anti-tubulin antibody. DES induced tetra- and octa-ploidy and DES monomethyl ether induced only tetraploidy at a much slower rate, whereas DES dimethyl ether was found to be completely inactive. Furthermore, DES was more active than its monomethyl ether in disturbing microtubule formation within cells. These results support the initial assumption that polyploidy is largely a consequence of the disturbed assembly of microtubules.

Biological Monitoring of Solar UV Radiation at 17 Sites in Asia, Europe and South America from 1999 to 2004

Abstract A small and robust dosimeter for determining the biologically effective dose of ambient UV radiation has been developed using UV-sensitive mutant spores of Bacillus subtilis strain TKJ6312. A membrane filter with four spots of the spores was snapped to a slide mount. The slide was wrapped and covered with two or more layers of polyethylene sheet to protect the sample from rain and snow and to reduce monthly-cumulative doses within the measurable range. From 1999, monthly data were collected at 17 sites for more than 1 year, and data for 4 to 6 consecutive years were obtained from 12 sites. Yearly total values of the spore inactivation dose (SID) ranged from 3200 at subarctic Oulu to 96 000 at tropical Denpasar, and the mean yearly values of SID exhibited an exponential dependence on latitude in both hemispheres with a doubling for about every 14 degrees of change. During the observation period, increasing trends of UV doses have been observed at all sites with more than 5 years of data available. Year-to-year variations at high and middle latitude sites are considered due mostly to climatic variation. At three tropical sites, negative correlations between the yearly doses and the column ozone amounts were observed. The results verified the applicability of spore dosimetry for global and long-time monitoring of solar UV radiation, in particular at tropical sites where no monitoring is taking place.

The sensitivity to DNA single strand breakage in mitochondria, but not in nuclei, of Chinese hamster V79 and variant cells correlates with their cellular sensitivity to hydrogen peroxide.

To investigate whether a relation exists between the level of DNA damage by and cytotoxicity of hydrogen peroxide, we measured the initial level of H2O2-induced nuclear and mitochondrial DNA single strand breaks in Chinese hamster V79 and H2O2-resistant variant cells (Hpr-4) with an alkaline elution technique and a quantitative Southern blot technique, respectively. The frequency of DNA single strand breaks in mitochondrial DNA induced by H2O2 was more than one hundred times that of nuclear DNA in the parent V79 cells. While a similar frequency of nuclear DNA single strand breaks was generated in V79 and Hpr-4 cells at an equidose of H2O2, a lower number of mitochondrial DNA single strand breaks were generated in Hpr-4 cells than in V79 cells by H2O2 in the range of 100 microM to 5 mM. The sensitivity to mitochondrial DNA single strand break-induction correlated with the cellular sensitivity to H2O2 in Chinese hamster V79 and variant cells.

Bimodal Pattern of Killing of Chinese Hamster V79 Variant Cells by Hydrogen Peroxide

To elucidate the mechanism of cytotoxicity of H2O2, we selected H2O2-resistant Chinese hamster V79 cells by single-step selection from a pool of spontaneous variants. The resistant cells showed bimodal sensitivity to H2O2 without exhibiting a significantly higher level of the detoxicating enzymes, catalase, glutathione peroxidase and superoxide dismutase. Mode-one and mode-two killing were observed at lower (< 300 microM) and higher (> 2 mM) H2O2 concentrations, respectively. Mode-one but not mode-two killing was prevented by iron chelators. Pretreatment with low concentrations of ascorbic acid preferentially enhanced the killing at higher H2O2 concentrations. These resistant cells were cross-resistant to t-butyl hydroperoxide and cumene hydroperoxide.

Relationship between the induction of mitotic gene conversion and the formation of thymine glycols in yeast S. cerevisiae treated with hydrogen peroxide

The effects of hydrogen peroxide on yeast Saccharomyces cerevisiae were assessed by measuring gene conversion at the trp 5 locus and the amount of thymine glycols in DNA using a monoclonal antibody specific to this base modification. Our results show that: (a) hydrogen peroxide-induced mitotic gene conversion in yeast strain D7M1 was dose-dependent in the low dose range where no toxicity was observed; (b) in the low dose range, the frequency of gene conversion depended on the temperature of the treatment, with more conversion at 25 degrees C than at 15 degrees C; (c) thymine glycols were induced in DNA in a dose-dependent manner following exposure of cells to up to 400 mM hydrogen peroxide; (d) there was little difference in the amount of thymine glycols formed in DNA when treatment occurred at either 25 degrees C or 15 degrees C.

The localization of DMPO spin adducts of .OH in endothelial cells exposed to hydrogen peroxide

Examination by electron spin resonance (ESR) spectroscopy revealed the localization of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) spin adducts of hydroxyl radicals (.OH) produced by bovine endothelial cells exposed to hydrogen peroxide. Addition of 10 mM chromium oxalate, a line-broadening agent, to the reaction mixture virtually abolished the signal of DMPO-OH spin adducts. Moreover, the spin adducts were recovered in the filtrated fraction of the cell suspension. We, therefore, concluded that the location of DMPO-OH due to .OH radicals produced by endothelial cells was extracellular. Contrastingly, the site of formation of DMPO-OH was confirmed to be intracellular by the effect of Desferal, an iron chelator, and the effect of poly(ethylene glycol), an extracellular scavenger of OH radicals, as previously reported. The DMPO-OH adducts in the cell suspension mixture were degraded by a cyanide sensitive pathway and they were apparently more unstable than in the extracellular fraction. The initial amount of DMPO-OH adducts formed in endothelial cells could potentially be monitored by the DMPO-OH signals in the extracellular reaction mixture better than those in the cell suspension mixture.

MITOCHONDRIAL BIOSYNTHESIS CONTROLS THE SENSITIVITY OF CHINESE HAMSTER CELLS TO HYDROGEN PEROXIDE

The mechanism of H2O2-resistance of Hpr-4, a variant of Chinese hamster V79 cells, was investigated. The effect of H2O2 on the mitochondria of the parental and Hpr-4 cells was compared. First, both biochemical and ultrastructural results showed that mitochondria in the parental cells were damaged by exposure to H2O2, while those in Hpr-4 cells recovered from the damage. Second, the H2O2-resistance of Hpr-4 cells was reversibly reduced or recovered by the addition or removal of inhibitors of mitochondrial biosynthesis, respectively. Third, the parental cells were auxotrophic to pyruvate after exposure to H2O2. Fourth, H2O2-sensitivity of the parental cells was also enhanced by the inhibition of mitochondrial biosynthesis. From these results, it was concluded that the mitochondria of Hpr-4 cells apparently had a greater resistance to H2O2 than those of the parental cells and that functional mitochondria were involved in the recovery of Chinese hamster V79 cells from H2O2-induced damage.

Repair of indirectly induced DNA damage in human skin fibroblasts treated with N-hydroxy-2-naphthylamine

The DNA lesions induced by active oxygen species generated from N-hydroxy-2-naphthylamine were quantitated by the alkaline elution technique as single-strand breaks using cultured human-skin fibroblasts. When cells were treated at 20 degrees C for 2 h with 0-25 microM carcinogen, the lesions increased biphasically with the concentration; the increase was slight below 10 microM while it was much larger and dose-dependent above this concentration. The dose response was similar for normal and xeroderma pigmentosum fibroblasts of complementation group A. There was no difference in the repair rate of single-strand breaks formed in these fibroblasts. The rates of repair of single strand breaks induced by N-hydroxy-2-naphthylamine and hydrogen peroxide were similar but slower than that of the repair of gamma-ray-induced single-strand breaks.

TYROSINE‐SENSITIZED PHOTODIMERIZATION OF THYMINE IN AQUEOUS SOLUTION

Abstract. Photodimerization of thymine in aqueous solution in the presence of tyrosine was studied with monochromatic UV irradiation. The total dimer formation was sensitized in the presence of tyrosine. The action spectrum of sensitized total dimer formation has a peak near 280 nm corresponding to the absorption maximum of tyrosine. Triplet quenchers reduced the sensitization substantially. It seems probable that tyrosine‐sensitized photodimerization of thymine occurred via triplet‐triplet energy transfer from tyrosine to thymine.