An inhibitor of potentially lethal damage (PLD) repair reduces the frequency of gamma-ray-induced mutations in cultured Chinese hamster V79 cells.

Cordycepin (3'-deoxyadenosine, 3'-dA) is an RNA antimetabolite and a radiosensitizer in cultured mammalian cells. In the present paper, the effects of 3'-dA on gamma-ray-induced lethality and 6-thioguanine (6TG)-resistant mutations in cultured Chinese hamster V79 cells were examined. 3'-dA had the effect of sensitizing the lethality induced by gamma-rays. The potentially lethal damage (PLD) repair produced by post-incubation of cells in Hanks' solution after gamma-irradiation was almost completely suppressed by 5 x 10(-5) M 3'-dA. When cells were irradiated with 10 Gy gamma-rays and incubated with 3'-dA for 5 h, the frequency of 6TG-resistant mutations induced by gamma-rays decreased to one-sixth of that of irradiated cells incubated without 3'-dA. The decrease in the frequency of gamma-ray-induced mutations was dependent on the length of incubation time with 3'-dA. It is suggested that the inhibition of PLD repair by 3'-dA may be that of error-prone repair.     

Peculiarities of the influence of chemical and physical factors on cytogenetic indices of root meristem in Pisum sativum L

The results of comparative analysis of cytogenetic effects of various chemical and physical factors on meristemic cells of field pea (Pisum sativum L.) are presented. Cytogenetic effects of aluminum, cadmium and selenium compounds as well as X-ray irradiation were studied. Cytogenetic indices were investigated for different doses of the above factors and peculiarities of their influence on the ratio of mitotic phases were analyzed. The increasing in the proportion of cells at the specific mitotic stage was revealed. There were anaphase stage for aluminum chloride, prophase stage for cadmium chloride, meta- and anaphase stages for sodium selenate, and telophase stage for X-rays. Investigated chemical elements may be ranged according to their ability to induce the aberrant anaphase frequency in the row: Se (from 3.75 x 10(-6) M) > A1 (from 3.86 x 10(-5) M) > Cd (from 8.44 x 10(-5) M). Maximal experimental dose of X-ray irradiation (9.03 x 10(-3) C/kg) induces the frequency of chromosome aberration similar to those induced by maximal experimental doses of aluminum and selenium (3.86 x 10(-4) M and 8.34 x 10(-6) M).     

2-[(Aminopropyl)amino] ethanethiol-mediated reductions in 60Co gamma-ray and fission-spectrum neutron-induced chromosome damage in V79 cells

The radioprotector 2-[aminopropyl)amino] ethanethiol (WR1065), which has been reported to reduce the cytotoxic and mutagenic effects of low LET radiation, was investigated for its ability to protect against low LET (60Co gamma ray) and high LET (fission-spectrum neutron)-induced chromosome damage in V79 cells. Cells were irradiated in G2 phase in the presence or absence of 4 mM WR1065 and were harvested and analyzed 2 h later for chromatid-type aberrations. Irradiation of G2-phase V79 cells in the presence of WR1065 resulted in a 30 to 50% reduction in the frequency of gamma-ray and neutron-induced chromatid-type breaks and exchanges. The effects were found only after exposures of greater than 200 cGy gamma-ray or 50 cGy neutron irradiation. The radioprotector was effective at reducing neutron-induced aberrations after exposures at dose rates of both 10 and 43 cGy/min. Thus the radioprotector WR1065 is an effective anti-clastogenic agent in V79 cells, protecting against both 60Co gamma-ray and fission-spectrum neutron-induced aberrations, when present during irradiation.     

Antimutagenesis by factors affecting DNA repair in bacteria

The term 'antimutagen' was originally used to describe an agent that reduces the apparent yield of spontaneous and/or induced mutations, regardless of the mechanisms involved. The 'antimutagens' include 'desmutagens' and 'bio-antimutagens'. In this article, our attention was focused on the bio-antimutagens affecting DNA repair in bacteria. Cobaltous chloride reduced the frequency of mutations in Escherichia coli induced by MNNG. The possibility that metal compound inhibits the growth of mutagen-treated cells was examined. The results clearly showed that the antimutagen surely reduces the mutation rate. The target of cobaltous chloride was found to be cellular factors including Rec A. Vanillin and cinnamaldehyde had strong antimutagenic activities against UV, 4NQO and AF-2. They stimulated Rec A-dependent recombination repair functions in the mutagen-treated cells. Among plant materials, tannins possess antimutagenic activity against UV-induced mutations in E. coli. It has been found that tannic acid stimulates the excision repair encoded by the uvrA gene thereby reducing the yield of mutants. Substances which are antimutagenic in bacterial systems also had antimutagenic activity in cultured mammalian cell systems. Vanillin reduced the frequency of mutagen-induced chromosomal aberrations.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.     

Comparative effects of 60Co gamma-rays and neon and helium ions on cycle duration and division probability of EMT 6 cells. A time-lapse cinematography study

Exponentially growing cultures of EMT 6 cells were irradiated in vitro with neon ions, helium ions or 60Co gamma-rays. Time-lapse cinematography allowed the determination, for individual cells, of cycle duration, success of the mitotic division and the age of the cell at the moment of irradiation. Irradiation induced a significant mitotic delay increasing proportionally with the delivered dose. Using mitotic delay as an endpoint, the r.b.e. for neon ions with respect to 60Co gamma-rays was 3.3 +/- 0.2 while for helium ions it was 1.2 +/- 0.1. Mitotic delay was greatest in those cells that had progressed furthest in their cycle at the time of irradiation. No significant mitotic delay was observed in the post-irradiation generation. Division probability was significantly reduced by irradiation both in the irradiated and in the post-irradiated generation. The reduction in division probability obtained with 3 Gy of neon ions was similar to that obtained after irradiation with 6 Gy of helium ions or 60Co gamma-rays.     

Modulation of radiosensitivity in Chinese hamster lung fibroblasts by cisplatin

The effects of cisplatin exposure time, concentration, and irradiation sequence on the sensitivity of Chinese hamster lung fibroblasts (V79) to gamma-ray exposure were examined. Based on clonogenic cell survival, the cisplatin concentrations corresponding to 50% cell survival (EC(50)) for exposure times of 1 h to 7 days followed a 2-phase exponential decay and ranged from 28.26 +/- 3.32 to 1.53 +/- 0.24 micromol/L, respectively. When cells were treated at EC(50) for exposures of less than 4 h and irradiated immediately, cisplatin inhibited the effect of radiation. Exposures of 4-6 h did not affect radiosensitivity. For exposures of 8-12 h, radiosensitization was observed, which disappeared at 14 h and reappeared for much longer cisplatin treatments. At the lowest achievable EC(50) (1.53 micromol/L), radiosensitization was observed if irradiation was delayed for 1-8 h. This enhancement in radiosensitivity disappeared for irradiation delays of 10-12 h, but reappeared when irradiation was delayed for 14-18 h. These data demonstrate that the mode of interaction between cisplatin and gamma-irradiation depends on the concentration and exposure time of cisplatin, as well as on the timing of irradiation after cisplatin administration. Consideration of changes in cell cycle kinetics may contribute to the improvement of treatment outcomes in adjuvant chemoradiotherapy involving cisplatin.     

Linear dose--response relationships after prolonged expression times in V-79 Chinese hamster cells.

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.     

Damage by gamma rays and heavy ions to superhelical structures of nuclear DNA

Chinese hamster cells (V79-4), human lymphocytes and mouse ascites cell were exposed to gamma-rays and heavy ions (4He and 12C). Sedimentation of complexes containing DNA was studied after cell lysis by centrifugation in a neutral sucrose gradient. The distinctions noted after irradiation with gamma-rays and heavy ions are consistent with the idea of the superhelical organization of DNA into discrete and membrane-bound compact units. According to the estimates made the diameter of these complexes was approximately 0.2 micron and DNA content, about 2 X 10(9) dalton.     

Radioprotection by caffeine pre- and post-treatment in the bone marrow chromosomes of mice given whole-body gamma-irradiation.

The effect of caffeine given as pre- and post-treatment in mice exposed to whole-body gamma-irradiation (1.5 Gy 60Co gamma-rays) was studied. The pre-treatment was either acute or chronic. The acute dose (5 mg/kg and 15 mg/kg body weight) was in the form of an injection given intraperitoneally, 30 min before irradiation. The chronic administration was in the form of caffeine solution (4.208 x 10(-3) M and 7.72 x 10(-4) M) contained in the drinking water that mice had had ad libitum access to instead of plain drinking water for 5 weeks prior to radiation exposure. The acute pre-treatment with caffeine reduced the radiation-induced frequency of chromosomal aberrations discernibly, whereas the chronic pre-treatment afforded a much more significant degree of radioprotection. The caffeine post-treatment (5 mg/kg and 15 mg body weight) was given in the form of an intraperitoneal injection to the mice immediately following whole-body gamma-irradiation. It is noted that both post-treatment concentrations of caffeine also significantly reduced the frequency of chromosomal aberrations induced by gamma-rays. These data are briefly discussed in terms of possible mechanistic considerations.     

Clastogenic effects of zinc chloride on human peripheral blood leucocytes in vitro

The clastogenic effects of three different concentrations of zinc chloride on human peripheral blood leucocytes were studied in vitro. The highest concentration (1.5 x 10(-3) M) was lethal after 48 and 72 h of culture and no blast cells were formed. The two lower concentrations (3.0 x 10(-4) M and 3.0 x 10(-5) M) significantly reduced the frequency of cell division, induced chromatid breaks and damaged cells in frequencies significantly higher than in control experiments maintained in sodium chloride and in distilled water.     

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.     

Mutagenicity of vinyl chloride, vinylidene chloride and chloroprene in V79 Chinese hamster cells.

The mutagenicity of vinyl chloride, vinylidene chloride (1,1-dichloroethylene) and chloroprene (2-chloro-1,3-butadiene) was tested in V79 Chinese hamster cells in the presence of a 15 000 x g liver supernatant from phenobarbitone-pre-treated rats and mice. Mutations in terms of 8-azaguanine and ouabain resistance were induced in a dose-related fasion by exposure to vapour of vinyl chloride in the presence of liver supernatant from phenobarbitone-pretreated rats. Vapours of vinylidene chloride and chloroprene induced a dose-related toxicity in the presence of liver supernatant from phenobarbitone-retreated rats, but these two compounds were not mutagenic in V79 Chinese hamster cells under the present assay conditions. The results are discussed with regard to the metabolic activation of the compounds and to the correlation with their carcinogenicity in man and experimental animals.     

Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes.

The genotoxicity of methyl mercury chloride (MMC, 0-25 x 10(-6) M) and dimethyl mercury (DMM, 0-434 x 10(-6) M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 x 10(-6) M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 x 10(-6) M and 1.73 x 10(-6) M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more clastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.     

Uptake of irradiated human low density lipoprotein by cultured Chinese hamster V79 cells

Specific binding and degradation of native and gamma-rays irradiated (100-2000 rad; 100 rad/min; 137Cs) human low density lipoprotein by Chinese hamster V79 cells and mouse peritoneal macrophage line, J774G were studied. Low density lipoproteins were labeled with 125I for studying the specific binding and subsequent degradation. The specific binding and degradation of irradiated 125I-low density lipoproteins (mixed with irradiated native lipoprotein) by Chinese hamster V79 cells are considerably reduced. The uptake depends on the concentration of thiobarbutaric acid-reactive products generated in the irradiated lipoproteins which in turn depends on the concentration of carotenoids. In contrast the rate of uptake of oxidized low density lipoproteins is enhanced by Chinese hamster macrophages. The alteration in the surface amino groups of apo-B of low density lipoprotein either due to direct damage of peptide bonds by gamma-rays or via interaction with lipid peroxides (generated in the core upon irradiation) are invoked as possible mechanisms for the reduction in specific binding and subsequent degradation by V79 cells.     

Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.     

用多引物PCR分析γ射线引起人外周血淋巴细胞hprt基因突变

正常人外周血用~（６０）Ｃｏ射线分别进行１～８Ｇｙ照射后,在含６－ＴＧ的培养基上克隆和筛选人淋巴细胞ｈｐｒｔ突变细胞。细胞的突变频率与γ射线的照射剂量呈正相关．获得４２个ｈｐｒｔ突变细胞株,用８对ｈｐｒｔ寡核苷酸引物进行多聚酶链反应（ＰＣＲ）从细胞粗提物中分别扩增ｈｐｒｔ各个外显子,分析突变细胞的ｈｐｒｔ基因突变,约６０％（２５／４２）的ｈｐｒｔ基因突变为基因缺失,其中１３个突变是ｈｐｒｔ基因全部缺失,而１２个是部分ｈｐｒｔ基因外显子缺失,约有４０％（１７／４２）的突变无明显的ＰＣＲ扩增变化．同时显示ｈｐｒｔ基因外显子的缺失突变与辐射剂量有关,实验结果提示,此方法有可能用于估计辐射剂量和进行辐射远后效果观察,进而揭示辐射致突的分子机理．     

Effect of copper ions on chromosome structural mutations caused by Cs1 37 gamma rays in moist seed cells of Crepis capillaris L]

When seeds of Crepis capillaris were germinated in solutions of Cu(NO3)2 (at 0.55-10(-5) M and 1.1-10(-5) M concentrations) the frequency of structural chromosome mutations induced by gamma-rays (137Cs) at 200 and 400 r doses was higher as compared to that induced in the absence of copper ions.