Influence of temperature on radiation-induced inhibition of DNA supercoiling.

The influence of subnormal temperatures (2, 15 and 28 degrees C) on the effects of radiation in MCF-7 cell cultures was studied using the fluorescent (halo) nucleoid assay. Increasing the propidium iodide (PI) concentration (0.5-7.5 microgram/ml PI) resulted in relaxation, i.e. in increasing nucleoid area; higher concentrations up to 50 microgram/ml caused rewinding that resulted in nucleoid contraction. Rewinding was inhibited by X irradiation (2, 4 and 8 Gy) in a dose-dependent way. Incubation at subnormal temperature did not influence the nucleoid area but did reduce radiation-induced inhibition of rewinding after 4 Gy. The low temperature (2 degrees C) during rather than prior to irradiation appeared to protect from radiation-induced inhibition of nucleoid rewinding. Decreased temperature during irradiation may change the conditions so as to reduce DNA- matrix damage induced by radiation.     

DNA mismatch repair mediates protection from mutagenesis induced by short-wave ultraviolet light

To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not significantly affect survival of these cells after UVC irradiation, UVC induced substantially more mutations in ES cells that lack the MutSalpha mismatch-recognizing heterodimer than in wild type ES cells. The global UVC-induced mutational spectra at Hprt and the distribution of most spectral mutational hotspots were found to be similar in mismatch-repair-deficient and wild type cells. However, at one predominant spectral hot spot for mutagenesis in wild type cells, the UVC-induced mutation frequency was not affected by the mismatch repair status. Together these data reveal a major role of mismatch repair in controlling mutagenesis induced by UVC light and may suggest the sequence context-dependent direct mismatch repair of misincorporations opposite UVC-induced pyrimidine dimers.     

Does Mutational Robustness Inhibit Extinction by Lethal Mutagenesis in Viral Populations?

Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Test of models for the sequence specificity of UV-induced mutations in mammalian cells

We have used mathematical modeling and statistical analysis to examine the correlation between UV-induced DNA damage and resulting base-substitution mutations in mammalian cells. The frequency and site specificity of UV-induced photoproducts in the supF gene of the pZ189 shuttle vector plasmid were compared with the frequency and site specificity of base-substitution mutations induced upon passage of the UV-irradiated vector in monkey cells. The hypothesis that the observed mutational spectrum is due to a preferential insertion of adenosine opposite UV photoproducts in the DNA template was found to best explain the mutational data. Models in which it was postulated that only (6-4) photoproducts, and not cyclobutane dimers, are mutagenic, or that the relative frequency of photoproduct formation does not influence mutation frequencies, fit the data much less well. This analysis demonstrates that molecular mechanisms of mutagenesis in mammalian cells can be deduced from mutational data obtained with a shuttle vector system.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin

Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01-0.1 microgram/ml, for 48 h and then treated with a high concentration (1.5 microgram/ml) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromosomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.     

Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells

CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.     

Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions. J. Bacteriol. 92:144-147. 1966.-T4r mutations were induced by ultraviolet irradiation of extracellular phage particles, using a phage mutant, v, which is particularly susceptible to photoreactivation. Most of the induced r mutations could be subsequently photoreversed intracellularly with white light. Ultraviolet irradiation induces both transitions and sign mutations, and both were susceptible to photoreversal. The results suggest that two very different types of mutational lesions may arise from a common type of photochemical lesion.     

MUTATION LOAD AND THE SURVIVAL OF SMALL POPULATIONS

Previous attempts to model the joint action of selection and mutation in finite populations have treated population size as being independent of the mutation load. However, the accumulation of deleterious mutations is expected to cause a gradual reduction in population size. Consequently, in small populations random genetic drift will progressively overpower selection making it easier to fix future mutations. This synergistic interaction, which we refer to as a mutational melt-down, ultimately leads to population extinction. For many conditions, the coefficient of variation of extinction time is less than 0.1, and for species that reproduce by binary fission, the expected extinction time is quite insensitive to population carrying capacity. These results are consistent with observations that many cultures of ciliated protozoans and vertebrate fibroblasts have characteristic extinction times. The model also predicts that clonal lineages are unlikely to survive more than 10⁴ to 10⁵ generations, which is consistent with existing data on parthenogenetic animals. Contrary to the usual view that Muller's ratchet does more damage when selection is weak, we show that the mean extinction time declines as mutations become more deleterious. Although very small sexual populations, such as self-fertilized lines, are subject to mutational meltdowns, recombination effectively eliminates the process when the effective population size exceeds a dozen or so. The concept of the effective mutation load is developed, and several procedures for estimating it are described. It is shown that this load can be reduced substantially when mutational effects are highly variable.     

Induction of neoplastic transformation in C3H/10T1/2 cells by 2.45-GHz microwaves and phorbol ester

C3H/10T1/2 cells were exposed to 2.45-GHz microwaves for 24 h and/or 1.5 Gy of 238-kVp X rays at 3.75 Gy/min. Transformation frequency and cell survival were measured with or without postirradiation addition of the tumor promoter tetradecanoyl-phorbol-13-acetate (TPA) at 0.1 microgram/ml. We previously reported (Carcinogenesis 6,859-864, 1985) an enhancement of transformation frequency when 10T1/2 cells exposed to a special sequence of microwaves and X rays were subsequently cultured in TPA. The same sequence of microwaves and X rays without promotion resulted in a transformation response similar to that induced by X rays alone. We now report statistically significant (at P greater than 0.999) enhancement of transformation response by TPA in cells exposed to 2.45-GHz microwaves (SAR = 4.4 W/kg). Microwaves alone had no effect on transformation. Plating efficiency and cell survival were not affected by TPA or microwave treatments.     

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in diploid human skin fibroblasts: an improved risk estimate in photochemotherapy

Cell killing and the induction of mutation were studied in dividing and non-dividing human skin fibroblasts as a result of treatment by 8-methoxypsoralen (8-MOP) and long-wave UV irradiation (UVA). The cytotoxic effect was highly dependent upon the duration of the UVA exposure. The frequency of mutations increased linearly with the UVA dose at concentrations of 10 and 0.25 microliter 8-MOP/ml, the latter representing the concentration in the skin during PUVA treatment. The number of mutations induced per unit dose (= per microgram 8-MOP/ml per joule UVA/m2) was calculated: for dividing cells this value was 3.3 X 10(-8) per cell and for non-dividing cells 0.6 X 10.8(-8) per cell. On the basis of these values the expected number of induced mutants in the human skin per session of photochemotherapy is 1.2 X 10(-5), and per 30 years of maintenance therapy 1.3 X 10(-2) per cell. A comparison was made between this frequency and the frequency to be expected from spontaneous mutation. In addition the significance of absence in patients of SCE induction by photochemotherapy is discussed.     

AMD3100 is a potent antagonist at CXCR4(R334X) , a hyperfunctional mutant chemokine receptor and cause of WHIM syndrome

WHIM is an acronym for a rare immunodeficiency syndrome (OMIM #193670) caused by autosomal dominant mutations truncating the C-terminus of the chemokine receptor CXC chemokine receptor 4 (CXCR4). WHIM mutations may potentiate CXCR4 signalling, suggesting that the United States Food and Drug Administration (FDA)-approved CXCR4 antagonist AnorMED3100 (AMD3100) (also known as Plerixafor) may be beneficial in WHIM syndrome. We have tested this at the preclinical level by comparing Chinese hamster ovary (CHO) and K562 cell lines matched for expression of recombinant wild-type CXCR4 (CXCR4(WT)) and the most common WHIM variant of CXCR4 (CXCR4(R334X)), as well as leucocytes from a WHIM patient with the CXCR4(R334X) mutation versus healthy controls. We found that CXCR4(R334X) mediated modestly increased signalling (~2-fold) in all functional assays tested, but strongly resisted ligand-dependent down-regulation. AMD3100 was equipotent and equieffective as an antagonist at CXCR4(R334X) and CXCR4(WT) . Together, our data provide further evidence that CXCR4(R334X) is a gain-of-function mutation, and support clinical evaluation of AMD3100 as mechanism-based treatment in patients with WHIM syndrome.     

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.     

Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast.

Previously we compared the mutational specificities of polychromatic UVB (285-320 nm) and UVC (254 nm) light in the SUP4-o gene of the yeast Saccharomyces cerevisiae. Striking similarities in the types and distributions of induced SUP4-o mutations were consistent with roles for cyclobutane dimers and pyrimidine(6-4)pyrimidone photoproducts in mutation induction by UVB. To assess the relative importance of cyclobutane dimers, we have now examined the effect of photoreactivation (PR), which specifically reverses these lesions, on UVB and UVC induction of SUP4-o mutations. PR reduced the frequencies of both UVB and UVC mutagenesis by approximately 75%. Collections of 138 and 158 SUP4-o mutants induced by treatment with UVB plus PR or UVC plus PR, respectively, were characterized by DNA sequencing and the results were compared to those for 208 UVB and 211 UVC-induced mutants analyzed earlier. PR decreased the frequency of UVB-induced G.C----A.T transitions by 85%, diminished the substitution frequencies at individual sites by 64% on average, and reduced the mutation frequencies at the five UVB hotspots by 87%. A more detailed examination revealed that the transition frequencies at the 3' base of 5'-TC-3' and 5'-CC-3' sequences were decreased by 90% and 72%, respectively. Finally, PR appeared to occur to the same extent on both the transcribed and non-transcribed strands of SUP4-o. Similar results were obtained for PR following UVC irradiation. Our findings indicate that cyclobutane dimers are responsible for the majority of UVB mutagenesis in yeast.     

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.     

大肠杆菌离子束诱变及rpoB基因两个新的利福平抗性位点鉴定

以60Co-γ射线辐照为参照体系,研究了低能氮离子诱发大肠杆菌利福平抗性突变。结果表明,低能氮离子注入具有损伤轻而突变率较高的特点。碱基置换型突变与其检出频率分析表明,CG→TA、GC→AT、AT→GC转换与AT→TA颠换为低能氮离子诱发大肠杆菌活体细胞内的高频突变,占检出总突变数的875% (77/88)。并鉴定出大肠杆菌rpoB基因中两个新的利福平抗性决定位点。位点一位于1551位鸟嘌呤脱氧核苷酸（dG）被胞嘧啶脱氧核苷酸（dC）取代,导致Gln517 (谷氨酰胺残基) 被His (组氨酸) 替代;位点二位于1692的胞嘧啶脱氧核苷酸（dC）被胸腺嘧啶脱氧核苷酸（dT）替代,导致Pro564 (脯氨酸残基) 被Leu (亮氨酸) 取代,使突变子产生抗性。其中位点一还未见报道,位点二的同义突变已有报道,但1692位点C到T的核苷酸突变并没有得到鉴定。     

Protective effect of curcumin on gamma-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on gamma-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The gamma-radiation at different doses (1, 2 and 4Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4Gy irradiation. Curcumin pretreatment (1, 5 and 10microg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1Gy irradiation all the concentrations of curcumin (1, 5 and 10microg/ml) significantly protected the lymphocytes from radiation damage. At 2Gy irradiation, 5 and 10microg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4Gy irradiation both 1 and 5microg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10microg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against gamma-radiation induced cellular damage.     

Induction of mutations in males of the fish Oryzias latipes at a specific locus after gamma-irradiation

We have studied frequencies of mutations induced at the b locus of the fish, Medaka Oryzias latipes, after gamma-irradiation. Homozygotes for the b locus have colorless melanophores whose phenotypic expression can be distinguished from that of the wild type. An advantage of the use of oviparous fish for detection of skin color mutations is that the mutant phenotype can be confirmed as early as 1.5 days after fertilization because of the transparent egg membrane of the embryo. Wild-type (B/B) male fish were exposed to 4.75 or 9.5 Gy of 137Cs gamma-rays at a dose rate of 0.95 Gy/min and then mated with the female testers (b/b). A total of 77,761 F1 offspring were examined for mutation and other abnormalities. In the control, we had 1 mutant among 22,068 offspring, resulting in a mutation rate of 4.53 X 10(-5)/locus/gamete. However, this mutant embryo died before hatching. Therefore, in an attempt to present specific-locus mutation frequencies in the fish, the frequencies of color mutants that survived more than 4 days after hatching were used as frequencies of viable mutants; (number of viable color mutants)/(number of hatched fry that survived more than 4 days after hatching). In the 4.75 Gy-irradiated group the viable mutant frequencies were 45.0 X 10(-5), 69.7 X 10(-5) and 0/locus/gamete, while exposure to 9.5 Gy resulted in mutation rates of 217 X 10(-5), 130 X 10(-5) and 8.06 X 10(-5), respectively, for sperm, spermatids and spermatogonia. In comparison with viable color mutant frequencies those of the total color mutants, which include such mutants as ones that died before hatching (defined as number of total color mutants/number of fertilized eggs minus number of early deaths), were considerably higher. For sperm, spermatids, and spermatogonia after exposure to 4.75 Gy, the frequencies were 1180 X 10(-5), 629 X 10(-5) and 9.90 X 10(-5)/locus/gamete, respectively, and in 9.5-Gy-irradiated fish, the frequencies were 1940 X 10(-5), 953 X 10(-5) and 55.5 X 10(-5). Although our data are incomplete, the present results were compared with mutation induction in mice. We concluded that the frequencies of viable color mutants in the fish can be compared with those in mice.