Mutation induction in cultured human cells after low-dose and low-dose-rate gamma-ray irradiation: detection by LOH analysis.

To study the genetic effects of low-doses and low-dose-rate ionizing radiation (IR), human lymphoblastoid TK6 cells were exposed to 30 mGy of gamma-rays at a dose-rate of 1.2 mGy/hr. The frequency of early mutations (EMs) in the thymidine kinase (TK) gene locus was determined to be 1.7 x 10(-6), or 1.9-fold higher than the level seen in unirradated controls. These mutations were analyzed with a loss of heterozygosity (LOH) detection system, a methodology which has been shown to be sensitive to the effects of radiation. Among the 15 EMs observed after IR exposure, 8 were small interstitial-deletion events restricted to the TK gene locus. However, this specific type of event was not found in unirradiated controls. Although these results were observed under the limited conditions, they strongly suggest that the LOH detection system can be used for estimating the genetic effects of a low-dose IR exposure delivered at a low-dose-rate.

Mutations induced by heavy charged particles.

The relative biological-effectiveness of radiation is increased when cells or tissue are exposed to densely ionizing (high-LET) radiation. A large number of studies focus on the following aspects of the biological effects of high-LET radiation: (i) basic understanding of radiation damage and repair; (ii) developing radiotherapy protocols for accelerated charged particles; and (iii) estimation of human risks from exposure to high-LET heavy charged particles. The increased lethal effectiveness (cell inactivation) of high-LET radiation contributes to new methods for using radiation therapy, but it is also necessary to study the enhanced mutagenic effect of high LET radiation, because higher frequencies of mutation can be expected to provide higher rates of carcinogenicity with human exposure. It is important to note that both measures of biological effectiveness (lethality and mutagenicity) depend on the quality of radiation, the dose, dose-rate effects, and the biological endpoints studied. This paper is intended to provide a review of current research on the mutagenic effects of high-LET radiation, and is organized into three sections. First, are descriptions of the induced mutations studied with various detection systems (section 1) because the detectable mutations induced by ionizing radiation, including heavy-ions, depend largely on the detection system used. Second is a discussion of the biological significance of the dependence of induced mutations on LET (section 2). This is related to the molecular nature of radiation lesions and to the repair mechanisms used to help cells recover from such damage. Finally, applications of mutation detection systems for studies in space (section 3) are described, in which the carcinogenic effects of space environmental radiation are considered.

Mutagenicity of gamma-radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk(+/-) mice.

The recently developed Tk(+/-) mouse detects in vivo somatic cell mutation in the endogenous, autosomal Tk gene. To evaluate the sensitivity of this model, we have treated Tk(+/-) mice with three agents that induce DNA damage by different mechanisms, and determined spleen lymphocyte mutant frequencies (MFs) in the autosomal Tk gene and in the X-linked Hprt gene. gamma-Radiation, which produces single- and double-strand breaks by nonspecific oxidative stress, efficiently increased Hprt MF, but not Tk MF. Mitomycin C, which produces bulky DNA monoadducts and crosslinks, was mutagenic in both the Hprt and Tk genes, but the response was greater in the Tk gene. An inhibitor of the ligase function of DNA topoisomerase II, etoposide, did not increase Hprt MF, and induced a small, but nonsignificant increase in Tk MF. Combined with previous data, the results indicate that the two genes are differentially sensitive to many agents, and that the Tk gene is more sensitive than the Hprt gene to some, but not all types of DNA damage.

Detection of genetic alterations induced by low-dose X rays: analysis of loss of heterozygosity for TK mutation in human lymphoblastoid cells.

To elucidate the genetic influence of low-dose ionizing radiation at the chromosome level, we exposed human lymphoblastoid TK6-20C cells to 10 cGy of X rays. The TK mutation frequency was 5.7 +/- 1.3 x 10(-6) at the background level and 6.9 +/- 2.8 x 10(-6) after X irradiation. Although this small increase was not statistically significant (P = 0.40), we applied multilocus analysis using 4 TK locus markers and 12 microsatellite loci spanning chromosome 17 for TK mutants exhibiting loss of heterozygosity (LOH). The analysis demonstrated a clear effect of low-dose ionizing radiation. We observed radiation-specific patterns in the extent of hemizygous LOH in 14 TK mutants among the 92 mutants analyzed. The deleted regions in these patterns were larger than they were in the control mutants, where those restricted to the TK locus. Surprisingly, the radiation-specific LOH patterns were not observed among the 110 nonirradiated TK mutants in this study. They were identified previously in TK6 cells exposed to 2 Gy of X rays. We consider these hemizygous LOH mutants to be a result of end-joining repair of X-ray-induced DNA double-strand breaks.

X-irradiation effects on thymidine kinase (TK): I. TK1 and 2 in normal and malignant cells.

The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells (Feinendegen et al. 1984, Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5-1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1-5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity.

The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1-24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5 cent-amino-5 cent-deoxythymidine (5 cent-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.

Radiation-induced changes in nucleotide metabolism of two colon cancer cell lines with different radiosensitivities.

PURPOSE: To investigate changes in nucleotide metabolism after irradiation. MATERIALS AND METHODS: HT29 and SW48 human colon carcinoma cells were exposed to 60Co gamma-rays at doses ranging from 0 to 7.5 Gy. At different times after irradiation, the activities of nine enzymes involved in nucleotide metabolism were measured, the levels of thymidine kinase and deoxycytidine kinase proteins were evaluated by Western blot, and cell-cycle kinetics were analysed by flow cytometry. RESULTS: Changes in enzyme activities concerned not purine but pyrimidine metabolism and essentially the salvage pathway for deoxyribonucleotide synthesis. They were greater in the less radiosensitive HT29 cells. The levels of thymidine kinase and deoxycytidine kinase proteins changed in parallel with their activities. The metabolic changes in irradiated cells did not seem to be due to S-phase transition and the pattern of enzyme activity changes was different from that observed in proliferative cells. CONCLUSIONS: Radiation-induced changes in the salvage pathway for pyrimidine deoxyribonucleotide synthesis were observed. These findings could be exploited in cancer therapy because higher enzyme activities after irradiation suggest that radiation exposure may render cells more sensitive to the drugs activated by these enzymes.

Characterization of mutations induced by 300 and 320 nm UV radiation in a rat fibroblast cell line.

The cytotoxic and mutagenic activities of monochromatic ultraviolet light (UV) at four wavelengths (254, 290, 300 and 320 nm) were determined using a rat fibroblast cell line CREF stably infected with a retroviral vector carrying the neo and HSV-tk markers. In this system, mutations can be positively detected as acyclovir-resistant colonies. Although the action spectra for these activities closely fit some of the previously reported spectra for photochemical DNA modifications, erythema, cell killing and mouse skin carcinogenesis, they diverge at 320 nm from the absorption spectrum for DNA and the action spectrum for bacterial inactivation and mutagenesis. Structural comparison of the HSV-tk mutants detected after irradiation with 300 and 320 nm UV revealed (1) CC dimers and C oligomers as predominant targets at both wavelengths; (2) increased incidence of relatively large deletions at 300 nm; and (3) greatly increased frequency of tandem double mutations at both wavelengths and of clustered multiple mutations at 320 nm. These results suggest the involvement of distinct mechanisms specifically operating, or becoming evident, in UV-mediated mutagenesis at these different wavelengths in mammalian cells.

Molecular mechanisms of spontaneous and induced loss of heterozygosity in human cells in vitro.

The human TK6 lymphoblast cell line is heteroallelic at the thymidine kinase (TK) locus, with one functional and one nonfunctional allele. Cells that have undergone loss of heterozygosity (LOH) at TK can be selected and cloned in an in vitro assay. In order to study the extent of LOH, we have analyzed a total of 166 thymidine kinase-deficient mutants that arose either spontaneously or following induction by X-ray or ethyl methane sulfonate (EMS) using DNA probes in and around the TK gene on chromosome 17. Two distinct groups of mutants with different doubling times were identified. Among slow-growth mutants, the predominant change for both spontaneous and induced mutants was LOH that generally extended through the entire TK gene to both proximal and distal markers on 17q. While the majority of both spontaneous and X-ray-induced normal-growth mutants showed LOH, this was considerably more localized in scale for X-ray-induced mutants, which rarely involved the distal marker. LOH was rare among EMS-induced normal-growth mutants. LOH was never observed with a 17p marker, indicating that nondisjunctional events were not involved in any of the mutant clones examined. Densitometric analysis of the LOH mutants indicated mitotic recombination was a likely mechanism in more than half the spontaneous LOH mutants in both groups, whereas most induced mutants appeared to arise from simple deletions.

T cell potentiation by low dose ionizing radiation: possible mechanisms.

The phenomenon of a stimulatory response induced by an exposure to a low dose of an otherwise toxic agent has been observed in a wide variety of organisms, ranging from the simplest prokaryotes to higher eukaryotes and with a spectrum of stimuli. This would suggest that the phenomenon is evolutionarily conserved and biologically important. However, we do not understand the mechanism responsible for the phenomenon, although it has been known for over 100 y. A reasonable assumption would be that adequate models and challenging paradigms are lacking to resolve this fundamental problem. Evidence is presented to show that the potentiation of T cell response by exposing them to single or multiple low doses of ionizing radiation is a feasible cellular model to understand the phenomenon. In addition, several possible mechanisms are discussed, including the participation of stress proteins and prostaglandins in stabilizing the signal transducing, transcriptional and translational machineries, and the possible role of a more efficient mechanism of DNA repair.

Modification of effects of radiation on thymidine kinase.

Thymidine kinase (TdR-K) and the incorporation of iododeoxyuridine (IUdR) into DNA of murine bone marrow cells are acutely and temporarily inhibited by low doses (0.01 Gy) of whole-body gamma-radiation with a maximal effect at 4 h after exposure and full recovery at 10 h. The inhibitory effect was totally abolished by whole-body exposure to a strong static magnetic field of 1.4T. The present investigation was designed to gain insight into the mechanism(s) underlying the inhibition of TdR-K activity and the incorporation of 125I-UdR by challenging the system with various pharmacological and biochemical means. To this end the response of TdR-K and 125I-UdR incorporation into DNA to the administration of actinomycin-D, cycloheximide, cysteamine, misonidazole and procaine hydrochloride as well as to dietary manipulations, i.e. vitamin E deficiency and enrichment of the diet by soya oil, and to changes in the glutathione levels were investigated in bone marrow cells of irradiated and sham-irradiated mice. Furthermore, the effect of various NaHCO3 concentrations on optimizing the radiation-induced inhibition of TdR-K was investigated under conditions of radiation, vitamin E deficiency and enrichment of the diet by soya oil. The data point to the involvement of the cellular radical-detoxification system in changing the activity of TdR-K, especially on the basis of the concurrent increase of glutathione concentration and decrease in TdR-K activity.

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation.

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.

In vivo enzyme control through a strong stationary magnetic field--the case of thymidine kinase in mouse bone marrow cells.

The influence of a strong homogeneous and stationary magnetic field (SMF) on the activity of the enzyme thymidine kinase (TdR-K) in bone marrow cells, and as a consequence of this on the incorporation of 125I-labelled 5-iodo-2-deoxyuridine (125IUdR) into DNA of mice and into isolated bone marrow cells in vitro, was assayed after exposure of immobilized mice. No effect could be elicited in moving mice, in cells in suspension or in enzyme in solution. The response depended on the body temperature during exposure: at 27 degrees C and 29 degrees C there was an increase and at 37 degrees C and a depression of enzyme activity. The TdR-K activity at low temperature increased with the field strength ranging from 0.2 to 1.4T. Thirty minutes were required for full expression of the effect at 1.4T; 5-10 min were needed after exposure for a return to base-line levels. Mice were given total-body irradiation at a dose of 0.1 Gy 137Cs gamma rays and then exposed immediately to a magnetic field at 1.4T for 30 min at a body temperature of 27 degrees C; gamma irradiation no longer inhibited the enzyme. Exposure to the magnetic field further removed from the time of gamma irradiation, did not negate the inhibitory effect of gamma irradiation. The observed responses to given challenges in this complex system support the hypothesis that the magnetic field affects TdR-K activity by way of a mediating structure, such as a membrane.