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Purpose: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora., Materials and Methods: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined., Results: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET)., Conclusions: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

Purpose: Lentil (Lens culinaris Medik.), being a self-pollinated crop with narrow genetic base, is an important target crop for mutation breeding experiments. The purpose of the investigation was to create, select and characterize unique mutations in inflorescence traits that have strong impact on lentil yield and yield stability., Materials and Methods: Healthy and uniform seeds (moisture 11.0%) of Lens culinaris Medik. cultivar Pant L 406 were irradiated with 100, 200, 300 and 400 Gy of gamma rays. The mutagenized populations were maintained up to mutant generation third (M 3 ) to screen for stable mutations in the inflorescence architecture of the lentil. The selected mutant mp 'Multipodding' trait, i.e. multiple pods per peduncle, was morphologically characterized and quantified in subsequent mutant generation fourth (M 4) ., Results: The morphological characterization of the 'multipodding' mutant (mp) revealed substantial morphological mutations were induced by the treatment of gamma rays. The estimation of yield per plant (g) between the mutant (mp) and parent cultivar Pant L 406 showed non-significant variation due to significant reduction in seed weight., Conclusions: The novel 'multipodding' (mp) mutant induced in the present study can play a key role in understanding the genetic network controlling legume inflorescence architecture and in genomics-assisted breeding for development of elite lentil cultivars.

Purpose: Current study was aimed to enhance the confidence of consumers as well as entrepreneurs towards food irradiation program., Materials and Methods: In this work, safety of high dose (25 kGy) irradiated meat samples (HDIMS) was ascertained by scoring mutation frequency through a long-term sub-culturing study in Escherichia coli MG1655 cells (ATCC 700926) up to 1500 generations (at 1%), 250 generations (at 5% and 10%) and human lymphoblast thymidine kinase heterozygote (TK6) cell line (ATCC CRL-8015) [at two gene loci, tk -/+ (thymidine kinase) and hprt + (Hypoxanthine Phosphoribosyltransferase)] up to 156 generations using goat meat sample. Also these samples were assayed at further radiation doses of 10, 45 and 70 kGy at 2% concentration (in cell line), and 1% (in E. coli). Study was also performed with other meat samples such as chicken, fishes (pomfret and rohu) and shrimps by carrying out limited long-term sub-culturing trials in human lymphoblast cell line. Mutation analysis was also carried out using a novel DPAR (Differential loss of Plasmid Antibiotic Resistance) assay followed by sequencing of tc R (tetracycline resistance) gene of pBR322 plasmid isolated from E. coli cells grown for 1500 generations on HDIMS medium and RAPD (Random Amplified Polymorphic DNA) analysis of the genome., Results and Conclusion: None of the assays exhibited any induced mutation when analyzed at regular time intervals. RAPD analysis also did not indicate any change in its nucleotide sequence, ruling out the occurrence of any silent mutation. Thus, the present findings report absence of mutagenic effect of high dose irradiated meat samples.

Purpose: To demonstrate the abscopal mutagenic effect of low-energy-ion irradiation in dormant plant seeds, and its dependence on the targeted portion of seeds., Materials and Methods: Arabidopsis thaliana-lines transgenic for b-glucuronidase (GUS) recombination substrates and A. thaliana RADiation54 (AtRAD54) promoter::GUS were adopted. The seeds were irradiated from four specialised orientations with 30 KeV (40)Ar(+) ions. The homologous recombination frequency (HRF) and the expression levels of the AtRAD54 genein non-irradiated aerial plants were measured. Moreover, several post-embryonic developments, such as growth of primary roots, differentiation of root hairs, and germination of seeds and growth of true leaves, were also analysed., Results: It was shown that low-energy-ion irradiation of seeds led to significant increases in HRF in the non-irradiated aerial parts of irradiated plants and the aerial parts of naïve plants from irradiated progenitors. The low-energy-ion irradiation was also shown to induce an elevated expression of AtRAD54 gene in aerial plants, and to inhibit the post-embryonic developments of seeds. Moreover, the changes in HRF, expression level of the AtRAD54 gene and post-embryonic developments depended largely on the orientation of seeds with regard to low-energy-ion irradiation; and the root apical meristem (RAM)-orientated irradiation exhibited the largest effects on all biological endpoints assayed here., Conclusions: Low-energy-ion irradiation can induce an abscopal mutagenic effect in dormant plant seeds, the extent of which depends greatly on the targeted portion of seeds.

Tikvah Alper's interest in science was broad, from scrapie to mammalian cells and cancer. Much of her own work focused on cell lethality, like that of many other radiobiologists, but this was natural because of the simplicity of the endpoint cell survival and its relevance to cancer therapy. Tikvah had broader interests, however, that included the effects of radiation on living systems in general like the induction of cancer and the cellular and molecular processes contributing to it. In this essay, some ideas are developed that lie in the mainstream of her interests. Starting with functional measures of the recovery or repair from radiation damage, a role for repair is illustrated in connection with mutagenesis and neoplastic transformation both discussed in the context of radiation-induced cancer. These topics are central to a model explaining the anomalous enhanced neoplastic transformation and cancer observed when low doses of a high-LET radiation are protracted in time. Under particular circumstances, the formalism of the model predicts application to protracted low-LET exposures as in the instance of repair-deficient target cells and sporadic breast cancer. The latter discussion leads to the proposal that the paradigm in current use for evaluating cancer risk should be broadened: from a simple dose-effect relation to one that includes cell kinetics (during protracted exposures), cell-cycle dependencies, and the influence of cellular repair or the lack thereof.

Exposure of E. coli AB1157 (a K-12 wild-type strain) to very large, daily X-ray doses has produced mutant strains resistant to both X-rays and UV photons. Results reported here are with KS0(160), the most resistant strain isolated thus far. Relative to its parent, KS0(160) is about 2.3 x more resistant to X-rays and about 2.2 x more resistant to ultraviolet photons (ratios of sensitivities in air). Two other characteristics of KS0(160) make its responses to X-rays different from those of AB1157: KS0(160) has an oxygen enhancement ratio of only 1.8 compared with 2.7 for its parent; glycerol reduces the sensitivity of AB1157 by about 75% (in air), but the maximum effect in KS0(160) is only a 49% reduction in response. P1 transduction experiments showed that all the acquired resistance in KS0(160) is lost when SOS repair activity is genetically blocked by an inserted lexAl, indicating that the mutation(s) associated with the acquired resistance in KS0(160) are in wild-type genes involved with induced DNA repair (i.e. SOS repair activity).

The Moolgavkar-Venzon-Knudson (MVK) two-mutation model of carcinogenesis is an analytical model that predicts the variation of cancer yield-rate with time, and with dose of a carcinogen. The model is biologically based, and assumes that a specific mutation in a stem-cell will increase its rate of proliferation compared with that of unmutated cells, so that a clone of pre-malignant cells develops; a second specific mutation in any one of these will make it malignant, and a cancer will start to grow. The model has been used in recent years to analyse a number of sets of epidemiological data on carcinogenesis. The purpose of this paper is to point to a problem in the use of this model for radiation-induced carcinogenesis, namely that ionizing radiation causes reproductive death of stem cells, which leads to regenerative division and hence a change in the number of stem-cells at risk. The possible effects of such changes on the predictions of the model are discussed. At low dose-rates of continuous or chronic irradiation and at low doses of acute irradiation, it is expected that pre-malignant cells will be killed along with the unmutated cells, and that the regenerative division of the surviving pre-malignant cells will restore the numbers of both stem cells and pre-malignant cells to what they would have been in the absence of cell killing; hence, no net effect of the tissue regeneration is expected. At high dose-rates, the initial delay in regenerative division and subsequent faster proliferation are expected to lead to an initial reduction in tumour yield-rate with time (compared with that predicted by the MVK model) followed by a faster increase. For acute irradiation, in the particular case of beta-particle irradiation of the skin, at high doses where there are practically no surviving cells in the irradiated area, repopulation by unirradiated cells from the margin is predicted to lead to a decrease in tumour yield-rate with dose. The predictions have been compared with published data on the induction of osteosarcoma in mouse by repeated injection of 89Sr, the induction of skin tumours in rat by acute and chronic irradiation with electrons, and the induction of skin tumours in mouse by acute irradiation with beta-particles. At low doses and dose-rates the basic MVK model fitted the data well. At higher doses and dose-rates the expected effects of tissue regeneration were observed qualitatively, although there were some discrepancies in detail; these are discussed.

The spectrum of deletion sizes in mutants of two human bladder carcinoma cell lines has been examined. The cell lines were MGH-U1 and a radiation-sensitive subline (U1-S40b) that has been developed in this laboratory. Three groups, each of 20-30 mutants at the hprt locus were investigated: arising spontaneously, or induced after exposure to 10 Gy gamma-radiation either at high dose-rate (2 Gy/min) or low dose-rate (0.01 Gy/min). Data on the mutation frequency of the two cell lines at low dose-rate were obtained to supplement previously published data at high dose-rate. The mutation frequency was lower in U1-S40b than in MGH-U1 both for high and low dose-rate irradiation. The presence of intact copies of each of the nine hprt exons was examined using multiplex PCR, supplemented by single-exon PCR. The incidence of small hprt mutations (i.e. leading to no change in the size of the PCR products) was the same for spontaneous mutations in the two cell lines; for radiation-induced mutants it was higher in U1-S40b. The incidence of total deletions (i.e. no positive exon amplification) was lower in U1-S40b both for high and low dose-rate irradiation. The results are consistent with the hypothesis that large deletions tend to lead to the loss of adjacent essential genes and thereby to the death of potential mutants.

An inverse dose-rate effect has sometimes been observed for mutagenesis in cells exposed to gamma-rays. We model such data quantitatively with the key assumption that the effect is caused in cycling cells by correlated variations in sensitivity across the cell cycle, for both mutation and killing. We quantify this approach using the LQR (linear-quadratic + resensitization) formalism, which describes the response to radiation of a heterogeneous cell population. This model is applied to an exponentially growing population. We compare its predictions with dose- and dose-rate dependent mutation data and show that it can well fit the observed inverse dose-rate effect, as well as providing an explanation of why inverse dose-rate effects have been seen in some experiments, but not in others. The actual values of the model parameters emerging from the analysis are reasonable in magnitude, based on their biological interpretations. We conclude that the LQR model can quantify cell-cycle redistribution effects without overparameterization, and that the data favour a correlation explanation of inverse dose-rate effects for mutagenesis by low-LET radiation. It is less clear that this explanation is appropriate to high-LET radiation-induced oncogenic transformation, although all potential explanations of inverse dose-rate effects predict that, at appropriately low doses, no dose-rate effects of any kind are expected.

Chronic exposure of V79 cells to 80 daily doses of 150 J/M2, 290-330-nm ultraviolet light (UVB) produced a mixed cell population that was found to be generally more resistant to cell killing by both UVB and UVC (254 nm) than the wild-type cells. Several subclones from this population were studied for their survival and mutation responses and then one was chosen for further characterization based on this data. The studies carried out on this subclone, designated N806, show that its spontaneous HPRT mutation rate is approximately 10 times higher than that of wild-type V79 cells and it is almost three times more mutable than the wild-type cells when both are induced by UVB or UVC. The mutation responses of N806 and MI2G cells to 50-kVp X-rays are different, but the N806 cells do not appear to be hypermutable as they are with UV. N806 cells are also moderately more resistant to the cytotoxic effects of UV radiation but are more sensitive than MI2G cells when exposed to X-rays. Assays to measure the removal of cyclobutane pyrimidine dimers (CPDs) and the incision step of nucleotide excision repair have revealed no detectable difference in the repair capacities of N806 and parental V79 cells. These results suggest that chronic, protracted UV irradiation may be able to induce a 'mutator phenotype' in a subpopulation of the progenitor cells.

B lymphocyte precursor cells are the target cells for the major subtype of paediatric cancer, acute lymphoblastic leukaemia. Using a murine IL-7-dependent clonogenic assay for normal B cell precursors as a model, we have investigated the sensitivity of these cells versus other normal and leukaemic haemopoietic cells to alpha-particle radiation. We find that B cell precursors are remarkably susceptible to the lethal effects of alpha-particles and have a very low probability of surviving a single alpha-track. B cell precursors are also very sensitive to the lethal effects of low LET X-rays. The mutation frequency in a marker gene (HPRT) does not, however, appear to be greater in B cell precursors that survive X-radiation than in other haemopoietic cells.

Tissue and locus specificity of mutation induction was studied in human mammary epithelial cells (HMEC). Primary HMEC from normal tissue, as well as immortalized HMEC (184B5) derived from normal HMEC, were cultured under identical conditions and exposed to 10 J/m2 ultraviolet (UV) radiation (254 nm peak wavelength), which produced approximately 50% mean survival in all cell strains and lines tested. UV radiation was found to induce mutations at the Na(+)-K+ ATPase locus as determined by ouabain-resistance in both normal and immortalized HMEC. Mutation frequencies measured in these cells following UV exposure were similar to those reported for human diploid fibroblasts. In addition, mutation induction was investigated at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in normal and immortalized HMEC. Induced mutations at the HPRT locus as determined by 6-thioguanine resistance in normal primary HMEC were not observed following UV radiation. In contrast, mutation induction was observed at this locus in UV-exposed immortalized HMEC.