Chemical modification of 5-[125I]iodo-2'-deoxyuridine toxicity in mammalian cells in vitro.

PURPOSE: To address the cytotoxic effects of DNA-incorporated (125)I in Chinese hamster V79 lung fibroblasts under various scavenging conditions. METHODS: The toxic effects of DNA-incorporated 5-[(125)I]iodo-2'-deoxyuridine ((125)IdUrd) were assessed by the colony-forming assay with cells incubated in medium containing serum and/or dimethyl sulphoxide (DMSO). Experiments were carried out at 0.3 or -135 degrees C. RESULTS: When (125)I decays were accumulated at 0.3 degrees C in 10% serum 0, 5 or 10% DMSO, no radioprotection was afforded by 5% DMSO, while the dose modification factor (DMF) for 10% DMSO was 2.0. For cells accumulating decays at 135 degrees C in the presence of 5 or 10% serum, DMSO was radioprotective (DMF= 1.8-1.9). D(0) obtained at each serum concentration correlated strongly (R=0.999) with the scavenging capacity of DMSO. Under these experimental conditions, 10% serum is approximately 3.6 times more protective than 5% serum. CONCLUSIONS: The contribution of indirect mechanisms to the toxicity of (125)I decaying within mammalian cell nuclear DNA can be demonstrated not only with DMSO, but also with the hydroxy radical scavengers present in serum.

Double-strand break yield following 125I decay--effects of DNA conformation.

The decay of iodine-125 (125I) is accompanied by the emission of low-energy electrons that dissipate most of their energy in approximately 10 nm from the decay site. In mammalian cells, the .OH generated by these electrons are also confined to a small volume. Iodine-125 is thus an excellent probe for assessing the radiobiologic effects produced by .OH in close proximity to the site of a decaying atom. We have compared in pUC19 plasmids (naked DNA) and in Chinese hamster V79 lung fibroblasts (chromatin) the modulation by the .OH scavenger dimethyl sulfoxide (DMSO) of 125I-induced DNA double-strand breaks (DSB). The data indicate that DMSO cannot protect plasmid DNA against DSB damage from 125I decaying within a few angstroms from DNA. However, DMSO attenuated DSB production in V79 cells following the decay of DNA-incorporated 125I, thus suggesting that chromatin structure fosters some DSB formation by indirect mechanism(s). DSB production depends on the environment and/or conformation of DNA. Consequently, current biophysical modeling of DNA damage that is based on naked and non-compacted DNA is inadequate for explaining radiobiologic effects at the cellular level.

Morphological transformation of C3H 10T1/2 cells by 99mTc-cardiolite.

UNLABELLED: The induction of in vitro morphological transformation in C3H 10T1/2 cells by 99mTc-Cardiolite (contents of Cardiolite kit [hexakis(2-methoxyisobutylisonitrile) and other components] plus (99m)Tc generator eluate) was examined. METHODS: Cells were grown for 48 h in the presence of 99mTc-Cardiolite or decayed 99mTc-Cardiolite (99mTc-Cardiolite after 1 wk of storage), and cell survival and transformation were assessed by the colony-forming and focus assays, respectively. X-ray was used as a reference for radiation effects, and 20-methylcholanthrene was used as a positive control for focus formation. RESULTS: Exposure of cells to 99mTc-Cardiolite results in a transformation frequency that is not significantly different from that induced by the volume equivalent of decayed 99mTc-Cardiolite. The number of foci per viable cell increases linearly from approximately 0.17 x 10(-4) in the untreated control to 1.7 x 10(-4) at 37 kBq/mL and 30 x 10(-4) at 1100 kBq/mL 99mTc-Cardiolite or its decayed 99mTc-Cardiolite volume equivalent. Furthermore, exposure of cells to low extracellular concentrations of 99mTc-Cardiolite or decayed 99mTc-Cardiolite (cell survival, > or =88%) induces an approximately 20-fold greater number of transformants per viable cell than that observed after 0.5 Gy x-irradiation, a dose that causes the same level of toxicity. CONCLUSION: Radioactive and decayed 99mTc-Cardiolite induce morphological transformation of C3H 10T1/2 cells in vitro. The underlying mechanism does not seem to be related to the radiation effects of decaying 99mTc but to chemical(s) present in the 99mTc-Cardiolite kit.

Toxicity of DNA-incorporated iodine-125: quantifying the direct and indirect effects.

To understand the biophysical mechanism(s) underlying the induction of cell death by the decay of the Auger electron emitter iodine-125 in DNA, Chinese hamster V79 lung fibroblasts were labeled with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU) for two doubling times and frozen and stored at -135 degrees C in the presence of 0.26-3.0 M dimethyl sulfoxide (DMSO), which acts simultaneously as a cryoprotector and a hydroxyl radical scavenger. After the accumulation of (125)I decays, the cells were defrosted and their survival was determined. Within the range of the number of decays examined (up to 470 disintegrations per cell), the survival curves are exponential. The dependence of the D(37) on DMSO concentration is triphasic and seems to reach a plateau at approximately 1.3 M. By extrapolating to infinite DMSO concentration, we estimate the D(37) for maximal hydroxyl radical scavenging to be 411 +/- 36 disintegrations per cell. To determine the D(37) in the absence of DMSO, we extrapolate the D(37) curve to zero concentration, and a D(37) of 54 +/- 5 disintegrations per cell is obtained. The maximal dose modification factor, calculated as the ratio of the D(37) at infinite DMSO concentration (i.e. direct effects only) to the D(37) at zero DMSO concentration (i.e. direct and indirect effects), is 7.6 +/- 1.0. By inference, approximately 90% of the radiotoxic effects of DNA-incorporated (125)I are due to indirect mechanisms.

Cytotoxicity of [125I]iodoHoechst 33342: contribution of scavengeable effects.

PURPOSE: The incubation of the DNA minor-groove binder [125I]iodoHoechst 33342 (125IH) with plasmid DNA leads to the production of one double-strand break (dsb) per decay, both in the presence and absence of dimethylsulfoxide (DMSO). In contrast, when 125I is incorporated into mammalian cell DNA as an iodinated pyrimidine base, DMSO decreases the dsb yield and enhances survival. Because these variations in radioprotective effects may be due either to the location of 125I vis-à-vis the DNA helix or to differences in DNA architecture, the toxicity of 125IH and its modification by DMSO were examined in mammalian cells. METHODS: Uptake and retention of 125IH in V79 cells were measured, and survival was determined after accumulation of 125I decays at 0.3 degrees C +/-10% DMSO. RESULTS: A linear-quadratic survival curve was obtained both in the absence [D37 = 114+/-36 decays/cell, alpha = (5.39 1.17) x10(-3) cell/decay] and presence [D37 = 211+/-65 decays/cell, alpha = (1.27+/-0.52) x10(-3) cell/decay] of DMSO. The dose modification factor for the linear component of the survival curve was 4.25+/-1.97, indicating the predominance of indirect mechanisms. This value is similar to that obtained with DNA-incorporated 125I (4.05+/-1.72) and for the initial slope (alpha) of 137Cs gamma-rays (4.43+/- 1.41). CONCLUSIONS: Cytotoxicity resulting from the decay of the Auger electron emitter 125I in the mammalian cell nucleus is caused mainly by indirect mechanisms.

Survival and DNA damage in Chinese hamster V79 cells exposed to alpha particles emitted by DNA-incorporated astatine-211.

Asynchronous Chinese hamster V79 lung fibroblasts were incubated at 37 degrees C for 30 min with the thymidine analog 5-[211At]astato-2'-deoxyuridine (211AtdU, exposure from DNA-incorporated activity) or with [211At]astatide (211At-, exposure from extracellular activity), and DNA-incorporated activity was determined. The 211AtdU content in cellular DNA increased as a function of extracellular concentration. Incorporation of 211At- was less than 1% of that of 211AtdU. After exposure, cells were frozen in the presence of 10% DMSO. One month later, survival was determined by the colony-forming assay, and DNA double-strand breaks (DSBs) were measured by the neutral elution method (pH 9.6). The survival curve for 211AtdU was biphasic (D37 = 2.8 decays per cell), reflecting killing of 211At-DNA-labeled cells and of unlabeled cells irradiated by 211At in neighboring labeled cells. The toxicity of 211At- decaying outside the cell (30-min exposure) was negligible. Analysis of the survival curve produced a D0 of 1.3 decays/cell for 211At-labeled cells. The yield of DSBs from the decay of DNA-incorporated 211At was compared with that from DNA-incorporated 125I. Each decay of 211At produced at least 10 times the number of DSBs as that obtained per 125I decay. The extreme radiotoxicity of DNA-incorporated 211AtdU seems to be associated with considerable damage to the mammalian cell genome.

Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: double-strand breaks.

We have examined whether nuclear DNA can be protected from double-strand breaks (DSBs) induced by decay of the Auger-electron-emitting radionuclide 125I. Decays were accumulated at 0.3 degrees C in Chinese hamster V79 cells suspended in isotonic buffer containing 0.1 M EDTA in the presence or absence of 10% dimethyl sulfoxide (DMSO). DSBs were measured by the neutral elution method (pH 9.6) and quantified as strand scission factors. DMSO was shown to protect DNA from DSBs caused by the decay of DNA-incorporated 125I. The dose modification factor (DMF) for this radionuclide decreases as a function of 125I decays (389 to 4,100 decays, DMF = 2.5 to 1.3). Extrapolation of the curve for the DMF indicates that at approximately 15,000 decays/cell, a DMF of 1 would be obtained. Experiments using large numbers of 125I decays confirmed these extrapolations. For induction of DSBs by 137Cs gamma rays, the DMF also decreases with dose (50 to 290 Gy, DMF = 2.7 to 1.5). However, extrapolation of the curve for the DMF indicates that protection does not cease at higher doses. The data show that, at the same level of damage, DMSO can protect against gamma-ray-induced DSBs 1.35-fold more efficiently than against DSBs caused by the decay of DNA-incorporated 125I. It appears that when 125I is incorporated into DNA, chromatin structure fosters some DSB formation by an indirect mechanism(s) and that more than one DSB is generated per decaying atom.

Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: clonogenic survival.

We have examined whether mammalian cells in vitro can be protected against the lethal effects of irradiation by Auger electrons emitted from DNA-incorporated 125I. Chinese hamster V79 lung fibroblasts were cultivated in the presence of 5-[125I]iodo-2'-deoxyuridine (125IdU) for 18 h and resuspended in ice-cold medium in the presence or absence of 10% dimethyl sulfoxide (DMSO). DNA-incorporated 125I activity was measured and the cells were plated for survival. A portion of the cell suspensions were also stored on ice to accumulate 125I decays for 6 to 48 h, after which the cells were plated to determine survival. Storage on ice up to 48 h without radioactivity reduced plating efficiency from 67 +/- 4% (SEM) to 20 +/- 1%. DMSO had a protective effect on colony formation, as the respective cloning efficiencies were 83 +/- 3% and 72 +/- 12% at 0 and 48 h. The survival curves for 125IdU-labeled cells are exponential with D0 = 36 +/- 2 decays per cell in the absence of DMSO and 195 +/- 20 decays per cell in the presence of DMSO. Thus the dose modification factor (DMF) at 37% survival for 10% DMSO is 5.4 +/- 0.6 for DNA-incorporated 125I. In reference experiments, a DMF of 2.5 +/- 0.8 was measured for cells irradiated with 137Cs gamma rays. These results indicate that the radiotoxicity of Auger electrons from 125I decay in mammalian cells is caused mainly by an indirect mechanism(s).