Irradiated Mammalian Cells Research Articles

Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

DNA Damage Produced by Ionizing Radiation in Mammalian Cells: Identities, Mechanisms of Formation, and Reparability

Publisher Summary This chapter provides a framework for understanding the way permanent heritable damage in cellular DNA results from exposure to ionizing radiation. If the processes that occur in the time between the initial radiation energy deposition and the formation of such damage can be described, then rationales can be developed for the modulation of radiation damage. For example, it may be possible to (1) devise a means of differentially modulating the amounts of radiation damage, for instance, in tumor versus normal tissue in radiotherapy, or (2) predict the effects of variations of several parameters on the biological consequences of a radiation dose. It has been concluded that the types and yields of damaged moieties produced in intracellular DNA by low LET ionizing radiation are consistent with the mechanisms of production that involve both OH radical attack and a direct ionization of the macromolecule. At the radiation-dose levels producing one lethal lesion per cell, the amounts of damage produced by ionizing radiation are several orders of magnitude lower than the amounts of damage required for equal kill by other agents. Individual damaged moieties are not biologically significant; they can easily be repaired using the undamaged DNA strand as a template. The lethal lesion produced in cellular DNA is a locally multiply damaged site.

Interactions of oxygen and sulfhydryls with free radicals in irradiated mammalian cells.

Studies on radiosensitization by oxygen and radioprotection by sulfhydryl-containing compounds have often directed attention to the importance of interactions of those two agents — oxygen and thiols — in determining radiation response. The so-called repair-fixation theory suggests radiation response is determined by a competition between damage fixation by oxidizing agents (e.g., oxygen) and damage repair by reducing species (e.g., sulfhydryls) 1,2: Open image in new window Open image in new window where T· represents an ionizing radiation-induced organic radical on a cellular “target” molecule, TH, such as DNA, and RSH is any thiol. This competition can be clearly demonstrated in simple chemical systems and in nucleic acids such as poly(A) and transforming DNA, and it has been shown that the damage fixation reaction is over 100 times faster than the H donation, chemical repair reaction (reviewed recently3). But the occurrence of this competition in mammalian cells is more difficult to demonstrate. This paper will review the current literature indicating the involvement of this chemical competition in determining radiation response in mammalian cells.

Formation of strand breaks in the DNA of?-irradiated chromatin

Strand breaks have been determined by sedimentation on sucrose gradients in the DNA of chromatin irradiated after isolation from Chinese hamster lung fibroblasts. The yields of double-strand and single-strand breaks are similar to those found in the DNA of irradiated mammalian cells. Irradiation of isolated chromatin in the presence of the radical scavenger tertiary butanol indicates that at least 65% of single-strand breaks and 56% of double-strand breaks can be attributed to the action of hydroxyl radicals. The results indicate the influence of chromosomal proteins in modifying radiation damage to DNA and suggest that the mechanisms for the induction of strand breaks in the DNA of isolated chromatin may be comparable to those operating in the intact cell.

DNA synthesis in irradiated mammalian cells.

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

The effects of benzamide ADP-ribosyl transferase inhibitors on cell survival and DNA strand-break repair in irradiated mammalian cells.

We have recently shown that 3-acetamidobenzamide (3-AAB), a highly effective inhibitor of ADP-ribosyl transferase (ADPRT), can act as a post-irradiation (electrons) sensitizer on the mouse lymphoma cell lines L5178Y R and S. We have now shown that this compound sensitizes human derived skin fibroblasts but to a lesser extent. Fibroblasts derived from normal, Friedreich's ataxia, and ataxia-telangiectasia individuals were equally sensitized by 3-AAB to electron radiation. 3-AAB was also effective in sensitizing the mouse lymphoma lines to fast neutron irradiation. In addition DNA strand break repair was retarded as had been found after electron irradiation. 3-Nitrobenzamide is structurally a potentially dual action radiation sensitizer with electron affinic and ADPRT inhibitory properties. It is a weaker inhibitor of ADPRT compared to 3-AAB, and results in a smaller sensitization of mouse lymphoma cells in air. However, a much greater sensitization is achieved in anoxia. This greater sensitization appears to be a synergistic rather than an additive combination of its electron affinic and ADPRT inhibitory properties.

Characterization of hydroxyl radical-induced damage after sparsely and densely ionizing irradiation.

The extent of hydroxyl radical mediated cell inactivation was measured for a variety of particle beams ranging from 8.5 Me V/u neon ions to 570 Me V/u argon ions. In general, the fraction of the total radiosensitivity caused by OH decreases from close to 60 per cent at low ionization density or low linear energy transfer (low LET) to close to 25 per cent at high LET for aerobically irradiated mammalian cells. The extent of OH induced cell lethality can be explained in terms of LET infinity only for low energy or low atomic number particles where fragmentations and complicated track structures do not contaminate the characteristic particle LET. For example, at a calculated LET infinity of 100 ke V/micron, the OH mediated fraction of the total radiation damage is about 25 per cent for low energy carbon but close to 40 per cent for high energy carbon ions. For low energy charged nuclei of approximately the same energy, as the 5.4-13.4 MeV/u He, Li, C and Ne ions in this report, there is a predictable diminution of the OH mediated effect with increasing LET infinity; however, the biological effect cannot be predicted accurately from calculated LET infinity values for high energy particle irradiation, nor indeed from a variety of low energy charged particles of quite different energies (incident velocities). This illustrates the unsuitability of using LET as a unifying parameter, except under specific circumstances. As more is learned about the energy deposition for energized charged particles in terms of track structure (core and penumbra), it may be possible to characterize the radiobiological data with a better physical parameter than LET infinity.

Repair of DNA breaks after irradiation in misonidazole or oxygen

Biphasic kinetics (half-times 7′ and 250′) adequately describe the aerobic repair of DNA breaks after irradiation of mammalian cells. The proportions repaired by the two components are affected by conditions prior to, during and after irradiation. DNA of cells irradiated in hypoxia have approximately twice as much of the damage which is repaired by the slow component as cells irradiated in air. If, instead of oxygen, misonidazole is present during hypoxic radiation, the repair resembles the repair of oxic damage more closely than repair of hypoxic damage. The biphasic nature of the repair curves is interpreted to be due to two classes of initial damage, proportions of which can be altered by sensitizers coach ac O 2 and misonidazole.

Oncogenic Transformation of Mouse BALB/3T3 Cells by Plutonium-238 Alpha Particles

The A31-11 mouse BALB/3T3 cell system was adapted for the study of malignant transformation induced by 5.3 MeV alpha particles from a specially constructed 238Pu source. The biologic effects were compared to those of 220 kVp X rays. The alpha-radiation survival curve showed a D0 of 70 rad and a relative biological effectiveness (RBE) of 3.5 at 50% survival. The transformation frequency increased exponentially with dose in the range examined (25-250 rad); the maximum RBE for the induction of transformation in growing cells was approximately 3. The RBE for alpha transformation in nonproliferating cells, however, appeared to be much higher; the yield of transformants among X-irradiated cells held in the stationary phase of growth for 6 to 220 hr after irradiation declined nearly 50-fold while no decrease occurred in alpha-irradiated cells. This finding supports the hypothesis that carcinogenic damage induced by high LET radiation in mammalian cells is inefficiently repaired compared with X-ray damage, and that the carcinogenic effect of exposures to high LET radiation may be simply cumulative in nature. It further suggests that the effective RBE for alpha radiation in nonproliferating cell populations in vivo may be much higher than one would predict based on measurements in dividing cells.

Irradiation of Mammalian Cells in the Presence of Diamide and Low Concentrations of Oxygen at Conventional and at Ultrahigh Dose Rates

The response of cultured CHO cells to ultrahigh-dose-rate radiation (approximately 10(9) Gy/sec) has been previously studied extensively using the thin-layer cell-handling technique developed in this laboratory. When the cells are equilibrated with a low concentration of oxygen, e.g., 0.44% O2, a breaking survival curve, due to radiolytic depletion of the oxygen, is observed. Hypoxic cells irradiated in the presence of the nitroimidazoles (e.g., misonidazole) are sensitized at ultrahigh dose rates in a dose-modifying manner, similar to that observed at conventional dose rates. These radiosensitizer compounds, if present in cells equilibrated with a low concentration of oxygen, prevent the breaking behavior of the survival curve, an observation believed to be due to the sensitizer interfering with the oxygen depletion process, leaving oxygen free to sensitize. Such experiments have recently been extended to studies with diamide, which, unlike the other sensitizers tested, acts primarily as a shoulder-modifying rather than a dose-modifying agent in hypoxic mammalian cells. These data indicate that diamide is active as a sensitizer at ultrahigh dose rates in a manner similar to that observed at conventional dose rates, and does modify the shape of the breaking survival curve observed with low concentrations of oxygen.

The Sequential Irradiation of Mammalian Cells with X Rays and Charged Particles of High LET

Chinese hamster V79 cells, synchronized in late-S phase, were irradiated with high-LET charged particles or X rays, or exposed sequentially to a single dose of charged particles followed by graded doses of X rays. The charged-particle irradiations consisted of deuterons (LET, 50 keV/microns) or 3He ions (96 or 160 keV/microns). The survival data obtained following the sequential irradiations show a synergistic effect compared with exposure to the low-LET radiation. An enhancement ratio is defined in order to quantify this effect. On the basis of this concept the present data show an enhanced interaction effect as the LET of the primary dose is increased or the size of this dose is increased. The data are also discussed in terms of a recent theoretical formulation which predicts synergism as a result of the interaction between the sublethal damage produced by the two radiations.

Rate of DNA synthesis in mammalian cells irradiated with ultravolet light: A model based on the variations in the rate of movement of the replication fork and in the number of active replicons

A model is proposed to describe the rate of DNA synthesis observed under certain conditions in UV irradiated mammalian cells. It is assumed that shortly after irradiation the rate of DNA synthesis drops mainly as a consequence of the drop in the rate of movement of the replication fork. This in turn, is due to a pause at the dimer for a limited length of time. Later on, a recovery in the rate of DNA synthesis occurs, and it is proposed that one of the parameters contributing to that is an increase in the number of active replicons. This simple model enables one to predict variations in the rate of DNA synthesis as a function both of UV dose and of time after irradiation.

Aphidicolin promotes repair of potentially lethal damage in irradiated mammalian cells synchronized in S-phase

Aphidicolin, an inhibitor of the α-polymerase in mammalian cells, at a concentration of 0.5 μg/ml, is shown to enable cells which are growing exponentially and synchronized in the S-phase of the cell cycle, to repair potentially lethal damage caused by exposure to either x-rays or UV-light. The drug holds cells up in the S-phase which may serve to allow time for repair and could prevent fixation of damage which may occur when the cells progress through the cell cycle. The possible involvement of α- and β-polymerase in repair of potentially lethal damage is discussed.

The Influence of Ionization Density on the DNA Synthetic Phase and Survival of Irradiated Mammalian Cells

The Influence of Ionization Density on the DNA Synthetic Phase and Survival of Irradiated Mammalian Cells

Chromosome Aberrations and Cell Survival in Irradiated Mammalian Cells

A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of X rays in early G1. Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-hr intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells (i.e., cell death) increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected.

The Influence of Ionization Density on the DNA Synthetic Phase and Survival of Irradiated Mammalian Cells

Depression of the DNA synthetic rate of exponentially growing V79 cells was transient with a dose-dependent maximum at 1 hour after exposure to sparsely or densely ionizing radiation. The dose-effect curves were biphasic for 241Am alpha-particles as well as for 60Co gamma-rays, being partly congruent if inhibition of DNA synthesis was expressed per S-phase cell. The lesions responsible caused a prolongation of the DNA synthetic period (S-phase) after sparsely ionizing X- or 60Co gamma-rays. However, no such effect was observed during the first 4 hours after exposure to densely ionizing alpha particles, peak pions and high LET neon ions. The effect was dose-rate independent. The inhibition of the DNA synthetic rate seems to be only partly related to survival.

Absence of correlation between DNA repair in ultraviolet irradiated mammalian cells and life span of the donor species.

The amounts of unscheduled DNA synthesis in fibroblasts originating from 34 species in 11 orders of mammals, after 10Jm-2 UV irradiation, were assayed by autoradiography. Genome size of the species was determined by cytophotometric measurements of DNA contents of the cultured cells and the ability of unscheduled DNA synthesis per unit amount of DNA was computed. There was no correlation between repair potentials and life span, denying the presence of the correlation suggested by Hart and Setlow on 7 species.

The wavelength dependence of 8-methoxypsoralen photosensitization of radiation-enhanced reactivation in a mammalian cell-virus system

The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus (Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet but not at others (240–289 nm). This same treatment induced RER in the near UV (302–370 nm) and the visible region (380–400 nm). An action spectrum for the photosensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.

Premature Chromosome Condensation Following X Irradiation of Mammalian Cells: Expression Time and Dose-Response

Premature chromosome condensation (PCC) in Chinese hamster ovary (CHO) cells following exposure to 300-kVp x rays was first detected in the mitosis that followed the second postirradiation S phase. Thus, cells irradiated in G1 first expressed PCC at the second postirradiation mitosis while cells irradiated in G2 did not express PCC until the third postirradiation mitosis. Cells irradiated in the S phase expressed PCC at the second postirradiation mitosis with a frequency that was related to the position of the cells in the S phase at the time of exposure, cells in the first half of the S phase (at the time of exposure) showing a higher frequency than cells positioned in the second half. Thus, DNA replication during the first postirradiation S phase may be involved in the processing of lesions that eventually give rise to PCC. For cells in G1 at the time of exposure, the D/sub o/ for PCC expression at the second postirradiation mitosis was around 825 rad, indicating that PCC may play only a minor role in x-ray-induced cell killing. Autoradiographic analysis indicated approximately 50% of the PCC patches scored were replicating DNA at the time condensation was attempted. Daughter cells derived from such cellsmore » would suffer loss of genetic material.« less

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET ENHANCED REACTIVATION IN A MAMMALIAN CELL‐VIRUS SYSTEM

Abstract— The effect of UV radiation in the wavelength region 230 nm to 302 nm on the ability of an irradiated mammalian cell to reactivate UV‐irradiated mammalian virus was tested. An action spectrum for radiation enhanced reactivation (RER) is presented. The shape of the action spectrum points to a combined nucleic acid‐protein target for UV radiation effects on this cellular parameter. An analysis of the results of others involving the biochemical and photobiological events involved in RER does not allow us to distinguish which macromolecule is the major contributor to this effect. Studies involving an analogous phenomenon in bacteria (Weigle reactivation) imply that RER and WR may involve similar mechanisms.