Kyomu Matsumoto

Suppression of 6-TG-resistant mutations in V79 cells and recessive spot formations in mice by vanillin☆

The antimutagenic effects of vanillin, anisaldehyde, cinnamaldehyde and coumarin were investigated in cultured Chinese hamster V79 cells in vitro. The frequencies of 6-TG-resistant mutations induced by UV or X-rays were decreased by treatment with each compound during the expression time. These decreases were not due to cytotoxic effects on cellular growth or killing effects on damaged cells. The antimutagenic effect of vanillin was also investigated in vivo in the mouse spot test using male PW and female C57BL/10 mice. Female mice were injected intraperitoneally with ethylnitrosourea (ENU) on the 10th day of pregnancy and received 3 successive oral administrations of vanillin. Administration of vanillin decreased the ENU-induced frequency of recessive carrier pups. These results indicate that vanillin acts as an antimutagen in mammalian cells both in vitro and in vivo.

Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by X-rays in mice

X-ray-induced chromosome aberrations were suppressed when vanillin, cinnamaldehyde, or p-anisaldehyde was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55-60% without toxicity of the test compounds to the bone marrow.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) induces gene mutations and inhibits metabolic cooperation in cultured Chinese hamster cells.

Investigations were carried out on 3‐chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX), a potent direct‐acting bacterial mutagen found in chlorinated tap water, for the potential to induce 6‐thioguanine (6‐TG) resistant mutations in cultured Chinese hamster V79 cells. The mutagenicity of MX was manifested when cells were treated with MX in Hanks balanced salt solution, but not in serum‐free Eagles minimal essential medium. A 12‐fold higher mutation frequency over control was obtained at 12.5 μg/ml MX. In addition, the inhibitory effect of MX on metabolic cooperation between cocultivated 6‐TG‐sensitive (6‐TG5) and ‐resistant (6‐TG1) V79 cells was investigated. Recovery of 6‐TG1 cells significantly increased at 0.8–2.0 μg/ml MX due to impaired intercellular communication with 6‐TGs cells. MX is believed to exert tumor‐promoting and mutagenic activity in mammalian cells.

In vivo anticlastogenic and antimutagenic effects of tannic acid in mice

The anticlastogenic effect of tannic acid was studied in vivo in the mouse micronucleus test. The frequencies of micronuclei induced by mitomycin C, ethyl nitrosourea (ENU) or 4-nitroquinoline 1-oxide in mouse bone marrow cells were decreased by the oral administration of tannic acid 6 h before the mutagen injection. The observed suppressing effect was not a reflection of a delay in the formation of micronuclei by the cytotoxic effect of tannic acid. The antimutagenic effect of tannic acid was also investigated in vivo in the mouse spot test using male PW and female C57BL/10 mice. Tannic acid was given orally to pregnant females 6 h before the intraperitoneal injection of ENU on the 10th day of pregnancy. The frequency of pups with recessive color spots induced by ENU was decreased by the administration of tannic acid. The observed decrease was not due to toxic effects on the embryo. These results indicate that tannic acid acts as an anticlastogen and antimutagen in vivo.

Suppressing effect of tannic acid on the frequencies of mutagen-induced sister-chromatid exchanges in mammalian cells

The effects of tannic acid (m-galloyl gallic acid) and 7 of its analogues on the frequencies of sister-chromatid exchanges (SCEs) were investigated in cultured Chinese hamster cells. SCEs induced by UV-light or mitomycin C (MMC) were suppressed by post-treatment with tannic acid and 5 of its analogues. These effects were independent of the extension of the cell cycle. The compounds which showed an SCE-suppressing effect have a common structure of 3 neighboring hydroxy or methoxy groups substituted on the phenyl group in benzoic acid or ester. These decreasing effects of tannic acid were observed in the G1 phase but not in the S or G2 phase of the cell cycle and a greater decline of the frequencies of UV-induced SCEs during liquid holding was seen in the presence of tannic acid. However, cells irradiated with X-rays were not influenced by tannic acid. In cells from a xeroderma pigmentosum (XP) patient, a Fanconis anemia (FA) patient, and a normal human embryo, MMC-induced SCEs were also decreased by post-treatment with tannic acid. Tannic acid reduced the SCE frequencies in UV-irradiated FA and normal human cells but not in UV-irradiated XP cells. Our results suggest that tannic acid modifies DNA-excision repair and that the decrease in the amount of unrepaired DNA damage might cause the reduction of induced SCEs.

Rotenone induces aneuploidy, polyploidy and endoreduplication in cultured Chinese hamster cells

The clastogenic potential of rotenone, an insecticide, was investigated in cultured Chinese hamster cells. Rotenone induced aneuploidy (hypodiploidy and hyperdiploidy), polyploidy, and endoreduplication, but not structural chromosome aberrations. The highest frequency of polyploidy and endoreduplication was 58.8% and 3.0%, respectively, when cells were treated with rotenone at 1.0 microgram/ml for 30 h.

Catalytic and non‐catalytic roles of DNA polymerase κ in the protection of human cells against genotoxic stresses

DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm‐6‐MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild‐type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non‐catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non‐catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed. Environ. Mol. Mutagen. 56:650–662, 2015.

Advances in the automated detection of metaphase chromosomes labeled with fluorescence dyes.

Applications of fluorescence in situ hybridization (FISH) for translocation studies and biological dosimetry would benefit substantially from reliable and efficient automatic detection of metaphase chromosomes labeled with fluorescent dyes. We replicated and evaluated a fluorescence metaphase finder previously developed at the Medical Research Council (MRC), Human Genetics Unit (Scotland) and at Lawrence Berkeley Laboratory (LBL; California). The MRC/LBL system seemed to detect nearly all of the metaphases on the test slides, but it presented an unacceptable number of false positives (about five false positives per one true positive). Furthermore, we determined that the system actually overcalled true detections by counting certain metaphase spreads twice (duplicates). Through modifications of the MRC/LBL system, we developed the Lawrence Livermore National Laboratory (LLNL) system, which minimizes the detection of duplicates, incorporates new detection features, uses a binary decision tree (BDT) for classification, and provides functionalities to improve scanning accuracy and improve the post-detection review. To test the new system, DAPI-stained preparations of metaphase chromosomes from blood lymphocytes of four unrelated donors were placed on slides in drops ranging from 7 mm to 20 mm in diameter. Drops contained between 5 and 200 scorable metaphases each. The LLNL system achieved approximately 90% detection of non-duplicated metaphases as verified by an expert cytogeneticist, with typically less than one false positive per every one true positive detected.

Enhancing effects of cinoxate and methyl sinapate on the frequencies of sister-chromatid exchanges and chromosome aberrations in cultured mammalian cells.

Sister-chromatid exchanges (SCEs) induced by mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or UV-light in cultured Chinese hamster ovary cells (CHO K-1 cells) were enhanced by cinoxate (2-ethoxyethyl p-methoxycinnamate) or methyl sinapate (methyl 3,5-dimethoxy 4-hydroxycinnamate). Both substances are cinnamate derivatives and cinoxate is commonly used as a cosmetic UV absorber. Methyl sinapate also increased the frequency of cells with chromosome aberrations in the CHO K-1 cells treated with MMC, 4NQO or UV. These increasing effects of methyl sinapate were critical in the G1 phase of the cell cycle and the decline of the frequencies of UV-induced SCEs and chromosome aberrations during liquid holding was not seen in the presence of methyl sinapate. Both compounds were, however, ineffective in cells treated with X-rays. In cells from a normal human embryo and from a xeroderma pigmentosum (XP) patient, MMC-induced SCEs were also increased by the post-treatment with methyl sinapate. The SCE frequencies in UV-irradiated normal human cells were elevated by methyl sinapate, but no SCE-enhancing effects were observed in UV-irradiated XP cells. Our results suggest that the test substances inhibit DNA excision repair and that the increase in the amount of unrepaired DNA damage might cause the enhancement of induced SCEs and chromosome aberrations.

Phases of the cell cycle sensitive to endoreduplication induction in CHO-K1 cells.

We investigated the sensitive phase of the cell cycle for endoreduplication induction by colchicine, vanadate, 4-nitroquinoline 1-oxide (4NQO), and hydrazine in Chinese hamster CHO-K1 cells, compared to the metaphase endoreduplication inducer rotenone. Treatment of asynchronous cultures and 5-bromodeoxyuridine (BrdU) labeling analysis showed that the detected endoreduplications originated from cells which had been treated with these inducers in S, G2, or metaphase. Exposure to synchronized metaphase cells revealed that the inducers did not lead metaphase cells to endoreduplicate. These results were markedly different from that of rotenone and suggested that the target for the induction of endoreduplication by the tested compounds existed between at least S and G2 phases.