Modification of cellular radiosensitivity by NO-synthase inhibitors

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect.We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.     

Mechanism of the radioprotective effect of cysteamine

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.     

mBAND analysis of chromosomal aberrations in human epithelial cells exposed to low- and high-LET radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.     

Modulation of bleomycin-induced mitotic recombination in yeast by the aminothiols cysteamine and WR-1065

The cancer chemotherapy drug bleomycin (BLM) is a potent inducer of genetic damage in a wide variety of assays. The radioprotectors cysteamine (CSM) and WR-1065 have been shown in previous studies to potentiate the induction of micronuclei and chromosome aberrations by BLM in Go human lymphocytes. By contrast, WR-1065 is reported to reduce the induction of hprt mutations by BLM in Chinese hamster cells. To elucidate the basis for these interactions, we examined the effects of CSM and WR-1065 on the induction of mitotic gene conversion by BLM in the yeast Saccharomyces cerevisiae. Treatment with BLM causes a dose-dependent increase in the frequency of mitotic gene conversion and gene mutations. Unlike its potentiation of BLM in G₀ lymphocytes, WR-1065 protected against the recombinagenicity of BLM in yeast. CSM was also strongly antirecombinagenic under some conditions., but the nature of the interaction depended strongly on the treatment conditions. Under hypoxic conditions, cysteamine protected against BLM, but under oxygenrich conditions CSM potentiated the genetic activity og BLM. The protective effect of aminothiols against BLM may be ascribed to the depletion of oxygen required for the activation of BLM and the processing of BLM-induced damage. Aminothiols may potentiatc the effect of BLM by acting as an electron source for the activation of BLM and/or by causing conformational alterations that make DNA more accessible tc BLM. The results indicate that aminothiols have a strong modulating influence on the genotoxicity of BLM in yeast as they do in other genetic assays. Moreover, the modulation differs markedly depending on physiological conditions. Thus, yeast assays help to explain why aminothiols have been observed to potentiate BLM in some genetic systems and to protect against it in others.     

Skewed X-chromosome inactivation in human miscarriages

The sex ratio in the first trimester of pregnancy shifts toward males due to increased elimination of female embryos. One reason for this phenomenon may be disruption of X chromosome inactivation. In this paper, we have analyzed the nature of the X chromosome inactivation in extraembryonic tissues of induced and spontaneous abortuses with 46,XX karyotype. Both equiprobable and asymmetric inactivation have been found in chorionic cytotrophoblast from spontaneous and induced abortuses. In the extraembryonic mesoderm of the control group of embryos, only equiprobable inactivation has been found, whereas this parameter was shifted in 15% of spontaneous abortions. The highest incidence of the selective inactivation of one of the parent homologues was found in the group with a lack of development of embryos and embryos from women with recurrent miscarriages. One of the reasons for the observed results can be compartmentalization of cells in the blastocyst leading to the nonrandom redistribution of cells and the predominance in the inner mass of cells with an active X chromosome with aberrations incompatible with normal embryonic development.     

Different modifications by vanillin in cytotoxicity and genetic changes induced by EMS and H2O2 in cultured Chinese hamster cells.

The modifying effects of vanillin on the cytotoxicity and 6-thioguanine (6TG)-resistant mutations induced by two different types of chemical mutagens, ethyl methanesulfonate (EMS) and hydrogen peroxide (H2O2), were examined using cultured Chinese hamster V79 cells. The effects of vanillin on H2O2-induced chromosome aberrations were also examined. Vanillin had a dose-dependent enhancing effect on EMS-induced cytotoxicity and 6TG-resistant mutations, when cells were simultaneously treated with vanillin. The post-treatment with vanillin during the mutation expression time of cells after treatment with EMS also showed an enhancement of the frequency of mutations induced by EMS. However, vanillin suppressed the cytotoxicity induced by H2O2 when cells were post-treated with vanillin after H2O2 treatment. Vanillin showed no change in the absence of activity of H2O2 to induce mutations. Post-treatment with vanillin also suppressed the chromosome aberrations induced by H2O2. The differential effects of vanillin were probably due to the quality of mutagen-induced DNA lesions and vanillin might influence at least two different kinds of cellular repair functions. The mechanisms by which vanillin enhances or suppresses chemical-induced cytotoxicity, mutations and chromosome aberrations are discussed.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells.

The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 micrograms/ml AsA, EMS-induced 6TG-resistant mutations were reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsA and EMS which had previously been incubated at 37.0 degrees C for 2 h. In pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.     

Concurrent detection of gene mutations and chromosome aberrations induced by five chemicals in a CHL/IU cell line incorporating a gpt shuttle vector

We previously established a transgenic Chinese hamster CHL/IU cell line, designated as KN63, for concurrent analysis of gene mutations and chromosome aberrations. The KN63 cell line contains copies of a shuttle vector with the Escherichia coli gpt gene as a mutational target in its chromosome. To evaluate the sensitivity of the cell line to various types of mutagens, methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), mitomycin C (MMC), vincristine sulfate (VIN) and C.I. basic red 9 hydrochloride (CIB) were assayed. KN63 cells were treated with each test chemical and gene mutations were detected in the gpt gene of the shuttle vector rescued from the KN63 cell genome into an E. coli host. Chromosome aberrations were concurrently evaluated by conventional metaphase analysis. MMS, ENU and MMC induced both gene mutations and structural chromosome aberrations in KN63 cells, with more efficient induction of the latter. VIN, a well-known aneugen, produced only numerical changes to chromosomes, while CIB was negative for both types of alteration. KN63 cells were as sensitive to MMS, ENU, MMC and VIN as Chinese hamster cell lines such as CHL, CHO and V79 cells. The characteristics of test chemicals indicated by this system should be useful for understanding endpoints in chemical mutagenesis.     

Incidence of chromosome aberrations in the bone marrow of monkeys and their progeny after exposure to aflatoxin B1

A mutagenic effect of aflatoxin B1 (AFB1) was studied in somatic cells of primates. It is shown that AFB1 elevates the chromosome aberration frequency in bone marrow cells of two monkey species: Macaca mulatta and Papio hamadryas. Genome mutations were found in these experimental primates simultaneously with chromosome aberrations. The AFB1-induced chromosome aberrations were revealed at least during two years of the study. The fact of the chromosome aberration frequency increase was established in Macaca babies born from females who received AFB1 in pregnancy.     

Aluminum induces chromosome aberrations in wheat root meristem cells]

The yield and pattern of chromosome structure aberrations in wheat seedlings treated with aluminum nitrate and aluminum sulfate at various concentrations have been determined by the anaphase method. Aluminum has a genotoxic effect causing genome, chromatid, and chromosome aberrations in apical root meristem cells. The relationship between the total yield of structural mutations and the aluminum concentration follows a bell-shaped curve. The mutagenic activity of aluminum nitrate peaks at 10(-3) mg/ml, which is twice as high as the permissible concentration limit (PCL) of aluminum in potable water. The maximum of the mutagenic activity of aluminum sulfate is observed at 5 x 10(-4) mg/ml, i.e., one PCL. Tap water boiled for 2 h in an aluminum vessel has virtually no genotoxic effect on wheat cells.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

The BIANCA model/code of radiation-induced cell death: application to human cells exposed to different radiation types

This paper presents a biophysical model of radiation-induced cell death, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), based on the assumption that some chromosome aberrations (dicentrics, rings, and large deletions, called “lethal aberrations”) lead to clonogenic inactivation. In turn, chromosome aberrations are assumed to derive from clustered, and thus severe, DNA lesions (called “cluster lesions,” or CL) interacting at the micrometer scale; the CL yield and the threshold distance governing CL interaction are the only model parameters. After a pilot study on V79 hamster cells exposed to protons and carbon ions, in the present work the model was extended and applied to AG1522 human cells exposed to photons, He ions, and heavier ions including carbon and neon. The agreement with experimental survival data taken from the literature supported the assumptions. In particular, the inactivation of AG1522 cells was explained by lethal aberrations not only for X-rays, as already reported by others, but also for the aforementioned radiation types. Furthermore, the results are consistent with the hypothesis that the critical initial lesions leading to cell death are DNA cluster lesions having yields in the order of ~2 CL Gy^?1 cell^?1 at low LET and ~20 CL Gy^?1 cell^?1 at high LET, and that the processing of these lesions is modulated by proximity effects at the micrometer scale related to interphase chromatin organization. The model was then applied to calculate the fraction of inactivated cells, as well as the yields of lethal aberrations and cluster lesions, as a function of LET; the results showed a maximum around 130 keV/μm, and such maximum was much higher for cluster lesions and lethal aberrations than for cell inactivation.     

The plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene increases the mutation frequency in Chinese hamster cells]

The induction of gene mutations and chromosome aberrations by the plasmid pEJ6.6 carrying the activated c-Ha-ras-1 oncogene from human bladder carcinoma was studied in cultured Chinese hamster cells. Both an increase in the frequency of gene mutations to 6-mercaptopurine resistance and of chromosome aberrations was observed after pEJ6.6 treatment as compared to control series (pBR322). Thus the results of experiments carried out show that the pEJ6.6 plasmid possesses a mutagenic activity.     

Genotoxicity assessment of new synthesized acridine derivative--3,6-diamino-10-methyl-9,10-dihydroacridine.

A new synthesized acridine derivative, 3,6-diamino-10-methyl-9, 10-dihydroacridine (AcrH), was tested for in vitro reverse mutations with Salmonella TA strains, chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes, and for in vivo chromosome aberrations in bone marrow of mice. Using the classic plate incorporation method, mutagenicity of AcrH in bacterial cells (TA97a, TA98, TA100 and TA102) was observed in the experiments performed with, and without, rat liver S9 metabolic activation. The reverse mutation assay showed no difference in mutagenic activity between AcrH and acriflavine (Acr(+)) in the test with TA97. The results of in vitro chromosome aberrations assay revealed potential clastogenicity. The test using macroculture of human lymphocytes induced mainly chromatid gaps. The experiments with human lymphocytes revealed SCE-inducing effect of AcrH and Acr(+). In an in vivo study, AcrH given intraperitoneally to Balb/c mice did not cause any significant increase in the percentage of cells with aberrations compared to the negative control.     

Cytogenetic activities of tobacco particulate matter (TPM) derived from a low to middle tar British cigarette

Tobacco particulate matter (TPM) derived from an experimental low to middle tar cigarette was tested for its cytogenetic activity upon a low passage number Chinese hamster pulmonary cell line. Examination of the mitotic profiles (after one cell cycle) revealed no interference by the agent with mitotic spindle formation and/or function. However, complete chromosomes (or parts of them) were seen to dislocate from the mitotic spindles. Such an event was probably the result of the chromosome aberrations, substantial numbers of which were observed in second division cells, or through a process of centromere inactivation. In second division cells there was a reduction in the number of diploid cells accompanied by an increase in both hypodiploidy and polyploidy and there was also a non-dose-related increase in endoreduplication. The results demonstrate that TPM was capable of inducing both structural and numerical chromosome aberrations in cultured mammalian cells.     

Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico

Data on 113,913 liveborn children from a hospital in Guadalajara, Jalisco (Mexico), were analysed for birth defects (BD); mutation rates were calculated for sporadic aneuploidy, chromosome aberrations and dominant gene mutations. The results showed a general incidence of 13.92 BD cases per 1000 liveborns, of which 1.64% were chromosomal abnormalities, 1.50% were aneuploid, 0.14% were structural chromosome aberrations and 3.23% were dominant gene mutations. The mutation rates were 8.20 x 10(-4) chromosomal abnormalities, 7.5 x 10(-4) aneuploidies, 7.0 x 10(-5) chromosome aberrations and 1.61 x 10(-3) dominant gene mutations/gamete/generation, respectively. The lethality rate was 15.32% of the liveborns with BD. The described findings estimate the incidence of new human mutants detected at birth in a sample of the Mexican population. They show that the rate for some aneuploidies are similar to those found in other populations previously reported in the literature but the rates of chromosome and dominant gene mutations were different.     

High Rates of Occurrence of Spontaneous Chromosome Aberrations in DROSOPHILA MELANOGASTER

Spontaneous mutations were accumulated for 40 generations in 140 unrelated second chromosomes with the standard gene arrangement. These were extracted from the same population by using the marked inversion technique, and the following findings were obtained: (1) In 42 out of the 140 chromosome lines, chromosome aberrations were detected by examining the salivary gland chromosomes: 40 paracentric and 15 pericentric inversions, 2 reciprocal translocations between the second and the third chromosomes, and 6 transpositions. (2) In 63 out of the 90 originally lethal-free lines, recessive lethal mutations occurred. (3) There were only 3 lines that acquired chromosome aberrations (inversions) with no lethal effects in the homozygous condition. (4) In a comparison of these results with those of the (CH), (PQ), and (RT) chromosomes in which no chromosome aberrations occurred after accumulating mutations for 22058 chromosome.generations (Yamaguchi and Mukai 1974), it was concluded that some of these 140 chromosomes carried a kind of mutator. (5) The frequency of mutator-carrying chromosome lines was estimated to be 0.66 on the basis of the distribution of the break-points on the chromosome lines and the frequency of lines that acquired neither recessive lethal mutations nor chromosome aberrations. Thus, the average number of breaks per mutator-carrying chromosome was estimated to be about 0.19/generation.On the basis of these estimates, the nature of the mutator factor was discussed.