Response of chronic hypoxic cells to low dose-rate irradiation.

PURPOSE: Compare the sensitivity of human cells in vitro to low dose-rate irradiation in air and in moderate hypoxia (4% O2). MATERIALS AND METHODS: Continuous low dose-rate beta-irradiation at a dose rate of 0.015 or 0.062 Gy/h was given to human T-47D breast cancer cells by incorporation of [3H] -labelled valine into cellular protein. Acute irradiation at a dose rate of 0.4 Gy/min was performed using [137Cs]gamma-irradiation. Cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia cabinet. RESULTS: When grown in ambient air with continuous irradiation, T-47D cells were able to continue growth for at least 23 weeks at a dose-rate of 0.015 Gy/h with a surviving fraction stabilized at around 60%. When the dose rate was increased to 0.062 Gy/h the cell culture died out after about 23 days (corresponding to about 22 Gy). When grown in an atmosphere with 4% O2 we surprisingly found that the continuously irradiated T-47D cells (0.015 Gy/h) were severely inhibited in their growth, and cell death became extensive after about 3 weeks while un-irradiated cells continued growth seemingly unaffected by this low oxygenation. Peri cellular oxygenation varied between 4% and below 0.1% over an ordinary passage due to diffusion-limitations through the 2 mm deep medium. Online O2-recordings over a whole passage showed that oxygen was more depleted in the irradiated compared to the un-irradiated cultures indicating increased respiration during irradiation. While cells growing attached to the bottom were inhibited and inactivated during irradiation it was found that cells attached high up in the neck region, i.e., having only a shallow layer of medium above them, survived and formed colonies. When cells cultivated in 4% O2 for 7 weeks were irradiated with acute doses of 137Cs gamma-rays, the radiosensitivity was the same as for cells cultivated in ambient air. CONCLUSION: Continuous irradiation with 0.015 Gy/h for several weeks results in a stronger inhibition for T-47D cells grown in an atmosphere with 4% as compared to 20% O2. The data indicate that this may be due to increased oxygen consumption resulting in more severe hypoxia in [3H]-incorporating compared to control (un-irradiated) cells.

Formates and dithionates: sensitive EPR-dosimeter materials for radiation therapy.

Polycrystalline formates and dithionates are promising materials for EPR dosimetry, as large yields of radiation induced stable radicals are formed with a linear dose response. Rapid spin relaxation rates were detected in many of the substances, indicating that a high microwave power can be applied during EPR acquisition in order to improve sensitivity. Different techniques used to further improve the sensitivity, such as the replacement of 7Li with 6Li or exchange of protons with deuterons in the corresponding crystalline matrices and metal ion doping are discussed. It is concluded that formates and dithionates may be up to 10 times as sensitive as L-alpha-alanine.

Radiation dose measurements with alanine/agarose gel and thin alanine films around a 192Ir brachytherapy source, using ESR spectroscopy.

Alanine/agarose gel and alanine films in stacks have been used for measurements of absorbed dose around an HDR 192Ir source in a vaginal cylinder-applicator, with and without a 180 degrees tungsten shield. The gel and the films were analysed by means of ESR spectroscopy and calibrated against an ion chamber in a 4 MV photon beam to obtain absolute dose values. The gel serves as both dosimeter and phantom material, and the thin (130 microm) films are used to achieve an improved spatial resolution in the dose estimations. Experimental values were compared with Monte Carlo simulations using two different codes. Results from the measurements generally agree with the simulations to within 5%, for both the alanine/agarose gel and the alanine films.

Free radical formation in X-irradiated crystals of 2'-deoxycytidine hydrochloride. Electron magnetic resonance studies at 10 K.

Single crystals of deoxycytidine hydrochloride (CdR.HCl) have been X-irradiated at 10 K with doses up to about 150 kGy and studied using 24 GHz (K-band) EPR, ENDOR and FSE spectroscopy. In this system, the cytosine base is protonated at the N3 position. Nine different radicals were characterized and identified. Three of these are ascribed to three versions of the one-electron reduced species, probably differing in their protonation state. Radicals formed by net hydrogen addition to the cytosine C5 and C6 positions were observed at 10 K. The hydrogen-abstraction radical at the deoxyribose C1' position most probably results from initial oxidation of the base. The remaining radical species are all localized to the sugar moiety, representing products formed by net hydrogen abstraction from three of the five available carbons of the deoxyribose sugar. The lack of base-centered oxidation products as well as the structures of the one-electron reduced species is rationalized by considering the specific proton donor-acceptor properties of this crystalline lattice in comparison with similar systems.

Measurement of dose rate at the interface of cell culture medium and glass dishes by means of ESR dosimetry using thin films of alanine.

Previous studies on human cervical cancer cells (NHIK 3025) have indicated that the cells, when X-irradiated in suspension, appeared to be more radiosensitive than when they were irradiated attached to glass dishes. However, this result depends on dosimetry, which is difficult in the situation where cells are attached to glass dishes due to backscattering electrons at the glass-liquid interface. Recently developed dosimetry that is based on detection of radiation-induced stable radicals in alanine and uses ESR spectroscopy offers a possibility for more relevant dosimetry at the glass-liquid interface than the previous estimates of doses based on Fricke dosimetry. Thin alanine films (>/=10 microm) were used to measure dose at the interface by irradiating the films while they were placed tightly against the bottom of dishes and covered with 1 mm of wax simulating the medium above cells. Fricke dosimetry was also performed, with different depths of Fricke solution in the dishes, to elucidate the contribution to the dose delivered by backscattering electrons at the glass-liquid interface. A dose rate of 1.9 Gy/min was measured with a thin layer (0.2-0.3 mm) of Fricke solution in petri dishes made of glass. However, this estimate appears to be too high, due to a contribution to dose by short-ranged electrons generated when the X rays passed through a steel lid 4.5 cm above the dishes. Dosimetry using alanine films resulted in dose rates of 1.15 and 0.87 Gy/min at the interfaces of glass-liquid and plastic- liquid, respectively. Hence there is a significant contribution to dose from backscattering electrons on dishes made of glass. The reason for our previous observation of a difference in radiosensitivity between cells irradiated in suspension and cells irradiated attached to glass appears to be a lack of accurate dosimetry at the glass-liquid interface.

Electron paramagnetic resonance and electron nuclear double resonance studies of X-irradiated crystals of cytosine hydrochloride. Part I: free radical formation at 10 K after high radiation doses.

Anhydrous single crystals of cytosine hydrochloride (protonated at N3) have been X-irradiated at 10 K and studied using K-band EPR, ENDOR and FSE spectroscopy. At least seven radicals were present at 10 K after X irradiation with a dose of about 150 kGy. Two different protonation states of the one-electron reduced cytosine cation were observed: an amino-protonated species (R1) and the pristine one-electron reduced species (R2) with zero net charge. Apparently three deprotonated versions of the one-electron oxidized cytosine cation were formed: the amino-deprotonated cation (R3), an N3-deprotonated cation (R4) and an N1-deprotonated cation (R5). Finally, two products formed by net hydrogen addition to the cytosine base were observed: a C5 hydrogen-addition radical (R6) and a C6 hydrogen-addition radical (R7). The crystalline lattice of cytosine hydrochloride is characterized in part by a cytosine base initially protonated at the N3-position, thus forming a cytosine base cation, and in part by an extended network of hydrogen bonding involving the chlorine anions. Proton transfer properties of pristine one-electron oxidation and reduction base products in this lattice are discussed and are suggested as explanations of the unusual multitude of positions for deprotonation of the one-electron oxidized species as well as for the two protonation states of the reduction product observed. The magnetic parameters for the amino-protonated species R1 agree well with those extracted from previous studies of cytosine derivatives in frozen solutions and in various glasses.

Radiation damage to DNA base pairs. II. Paramagnetic resonance studies of 1-methyluracil x 9-ethyladenine complex crystals X-irradiated at 10 K.

Single crystals of the co-crystalline complex of 1-methyluracil and 9-ethyladenine were X-irradiated and studied using EPR, ENDOR and FSE spectroscopic techniques at 10 K. All together seven radicals were identified, and experimental evidence for at least one more species, as well as for a very low population of radical pairs, is available. Oxidation and reduction products appear to be stabilized at both base constituents of the pair. Of the 1-methyluracil moiety, the product formed by net hydrogen abstraction from the methyl group was observed, together with the 1-methyluracil anion and the 1-methyluracil-5-yl radical. From the 9-ethyladenine moiety, the N3-protonated 9-ethyladenine anion is stabilized. In addition, the 9-ethyladenine cation as well as traces of the amino-deprotonated cation were observed, together with the C8-H hydrogen adduct. The presence of oxidation and reduction products in each of the two bases may indicate that negligible energy transfer takes place between them. This behavior is different from that observed in the similar pair of 1-methylthymine-9-methyladenine. There also seems to be minor proton exchange between the two stacks of molecules: Interbase protonation-deprotonation channeled through the hydrogen-bonding scheme seems to be almost completely suppressed.

Electron spin resonance and electron nuclear double resonance study of X-irradiated deoxyadenosine: proton transfer behavior of primary ionic radicals.

A study of deoxyadenosine crystals (anhydrous form) after irradiation at 10 K found four base-centered radicals and one sugar-centered radical. Radical R1, thermally stable to about 100 K and photobleachable easily with white light, was the product of deprotonation at the amino group by the primary radical cation. Radical R2, also thermally stable to about 100 K, was the product of protonation at N3 of the primary radical anion. Radical R3, stable to about 170 K, was centered in the deoxyribose moiety and evidently was the result of net hydrogen abstraction from C4'. Radicals R4 and R5 were the C2 and C8 H-addition products with couplings typical of those species. Both R4 and R5 were formed at 10 K and were stable at room temperature. The behavior of R1 in several systems provides additional evidence for significant involvement of the hydrogen-bonding environment in controlling the stabilization (or formation) of radicals resulting directly from ionization, as described previously (Radiat. Res. 131, 272-284, 1992). From comparison of amino-group hydrogen-bonding environments in which radicals with the structure of R1 were stabilized, we conclude that oxygen atoms as proton acceptors are important in permitting the charge and spin separation necessary for radical stabilization. In particular, oxygens of ROH structures seem most efficient by readily permitting a multi-proton shuffle through a mechanism amounting to proton exchange. The collective results show that stabilization of these products is unlikely unless the charge and spin can be separated by at least one intervening molecule.

Radiation damage to DNA base pairs. I. Electron paramagnetic resonance and electron nuclear double resonance study of single crystals of the complex 1-methylthymine.9-methyladenine X-irradiated at 10K.

Single crystals of the complex 1-methylthymine.9-methyl-adenine were X -irradiated at 10 and at 65 K and studied in the temperature range 10 to 290 K using K-band EPR, ENDOR and field-swept ENDOR (FSE) techniques. The EPR and ENDOR spectra are dominated by two major and four minor resonances. The two major resonances are: MTMA1, the well-known radical formed by net hydrogen abstraction fr om the CS methyl group of the thymine moiety, and MTMA2, the radical formed by net hydrogen abstraction from the N1 methyl group of the thymine moiety. The latter product has not been observed previously in any 1-methylthymine derivative. The four minor resonances are: MTMA3, the anion of 1-methylthymine, possibly protonated at the O4 position; MTMA4, the well-known species formed by net hydrogen addition to C6 of the thymine moiety; MTMA5, the species formed by net hydrogen addition to C2 of the adenine moiety; and MTMA6, the species formed by net hydrogen addition to C8 of the adenine moiety. Radical MTMA3, the O4-protonated thymine anion, was clearly detected at 10 K, but upon thermal annealing at 40 K the lines began to disappear. In crystals irradiated at 65 K MTMA3 was only weakly present. Radical MTMA2 decayed at about 250 K with no detectable successor, and radical MTMA5 disappeared at about 180 K. It was not possible to learn from the d ata if MTMA5 transformed into MTMA6. The radical distribution in the 1-methylthymine.9-methyladenine crystal system is different from that in crystals of the individual components. Reasons for this behavior are discussed in light of the hydrogen bonding schemes and molecular stacking interactions in each of the crystals. An important feature is the concept of excited-state transfer from the adenine to the thymine moiety, followed by dehydrogenation at the thymine Nl-methyl group, the mechanism resulting in radical MTMA2.

Free radical formation in single crystals of 9-methyladenine X-irradiated at 10 K. An electron paramagnetic resonance and electron nuclear double resonance study.

Single crystals of 9-methyladenine were X-irradiated at 10 K and at 65 K and were studied using K-band EPR, ENDOR and field-swept ENDOR (FSE) techniques in the temperature range 10 K to 290 K. Three major radicals are stabilized in 9-methyladenine at 10 K. These are: MA1, the adenine anion, probably protonated at N3; MA2, the species formed by net hydrogen abstraction from the 9-methyl group; and MA3, the radical formed by net hydrogen addition to C8 of the adenine moiety. Radical MA1 decayed at about 80 K, possibly into the C2 H adduct (MA4). The other two species (MA2, MA3) were stable at room temperature. A fifth radical species was clearly present in the EPR spectra at 10 K but was not detectable by ENDOR. This species, which decayed above 200 K (possibly into MA3), remains unidentified. The radical population at room temperature is as described by previous authors. The mechanisms for radical formation in 9-methyladenine are discussed in light of the hydrogen bonding scheme and molecular stacking interactions.

Electron paramagnetic resonance of X-irradiated sodium and potassium salts of glucose-1-phosphate. Identification of PO3(2-) radicals at room temperature.

Single crystals of the disodium and dipotassium salt of glucose-1-phosphate, X-irradiated at 80 K or at 280 K, show the presence of PO3(2-) radicals at 295 K, formed by scission of the phosphate-ester bond at the phosphate side. The 31P hyperfine coupling constants were measured using X- and Q-band EPR spectroscopy. Typical values for these coupling constants are a magnitude of = 68 mT and a perpendicular = 53 mT. The g values were almost isotropic, slightly smaller than that of the free electron spin (g = 2.0023). There is no substantial reorientation of the phosphate group in the crystalline lattice upon radical formation. Directly after irradiation at 77 K the PO3(2-) radicals are not present, but their characteristic resonance grows in upon thermal annealing of the crystals. The radicals are probably formed from a carbon-centered radical precursor by secondary reactions resulting in the loss of the phosphate group, leaving a (diamagnetic) modified carbohydrate molecule behind. The alternative process of reductive cleavage of the phosphate-ester bond by electrons released from traps in the crystal upon thermal annealing is considered less likely. A second phosphate-centered species, with a magnitude of about 21 mT and a perpendicular about 15 mT, was detected in the dipotassium salt of glucose-1-phosphate only. Possible structures for this species are discussed.

ESR and ENDOR study of single crystals of deoxyadenosine monohydrate X-irradiated at 10 K.

Five free radicals have been detected by detailed ESR/ENDOR experiments on single crystals of deoxyadenosine monohydrate (AdRm), X-irradiated and observed at 10 K. In a previous study of adenosine (Radiat. Res. 117, 367-378, 1989), only the anion (protonated at N3) and the cation (deprotonated at the exocyclic NH2) were detected at 10 K. In AdRm, Radical R1 is the N3-protonated anion, similar to that observed previously in adenosine. Radical R3 is a C5' hydroxyalkyl radical formed by net H-abstraction from C5'. A second sugar radical is formed by net C1' H-abstraction. Two other base radicals observed in AdRm at 10 K are the C2 and C8 H-addition radicals. The C2 H-addition radical (Radical R5) exhibits inequivalent methylene hydrogen couplings of 5.43 and 3.29 mT, while in the C8 H-addition radical (Radical R6) the couplings are somewhat more equivalent (3.63 and 4.17 mT). No link between RAdical R1 and the H-addition radicals has been observed. The reduced base appears to protonate rapidly even at 10 K, while at the same time both H-addition radicals are clearly present. On warming, Radical R1 appears to decay at about 80 K with no apparent successor. Although no base cation was stabilized in AdRm at 10 K, it is interesting to note that Radicals R3 and R4 could both be formed as the result of deprotonation of primary oxidation products.

Ionization of adenine derivatives: EPR and ENDOR studies of X-irradiated adenine.HCl.1/2H2O and adenosine.HCl.

Following X irradiation of adenine.HCl.H2O at 10 K, evidence for five distinct radical products was present in the EPR/ENDOR. (In both adenine.HCl.1/2H2O and adenosine.HCl, the adenine base is present in a cationic form as it is protonated at N1). From ENDOR data, radical R1, stable at temperatures up to 250 K, was identified as the product of net hydrogen loss from N1. This product, evidently formed by electron loss followed by proton loss, is equivalent to the radical cation of the neutral adenine base. Radical R2, unstable at temperatures above 60 K, was identified as the product of net hydrogen addition to N3, and evidently formed by electron addition followed by proton addition. Radicals R3-R5 could not be identified with certainty. Similar treatment of adenosine.HCl provided evidence for six identifiable radical products. Radical R6, stable to ca. 150 K, was identified as the result of net hydrogen loss from the amino group, and evidently was the product of electron loss followed by proton loss. Radical R7 was tentatively identified as the product of net hydrogen addition to C4 of the adenine base. Radical R8 was found to be the product of net hydrogen addition to C2 of the adenine base, and R9 was the product of net hydrogen addition to C8. Radical R10 was identified as the product of net hydrogen abstraction from C1' of the ribose, and R11 was an alkoxy radical formed from the ribose. With the exception of R11, all products were also found following irradiation at 65 K. Only radical R8 and R9 were stable at room temperature. Most notable is the different deprotonation behavior of the primary electron-loss products (radical R1 vs. R6) and the different protonation behavior of the primary electron-gain products (radical R2 vs. no similar product in adenosine.HCl). The major structural difference in the two crystals is the electrostatic environment of the adenine base. Therefore, this study provides further evidence that environmental influences are important in determining proton transfer processes.

On the proton transfer behavior of the primary oxidation product in irradiated DNA.

Results are surveyed from investigations of six adenine and six guanine crystal systems X-irradiated and studied at temperatures near that of liquid helium (T less than 10 K). Basic conclusions from the overall results are that the primary oxidation products deprotonate rapidly, and that the specific site of deprotonation is environment-dependent. The following systematic behaviors were identified: (1) in no case was there deprotonation at a site hydrogen-bonded to a halide ion (three examples with guanine and two with adenine); (2) likewise, in no case was there deprotonation at a site hydrogen-bonded to a phosphate group (three examples, all with guanine); (3) in no case was there deprotonation at a site involving an greater than N-H . . . N less than hydrogen bond; (4) in all cases except one, the site of net deprotonation involved greater than N-H . . . O hydrogen bonds. In the remaining case, the leaving proton was uninvolved in hydrogen bonding. A review of results obtained previously from DNA leads to the conclusion that the actual protonation states of DNA oxidation products are unknown at the present time. The results presented here predict with high probability that such DNA products also will deprotonate, but the environment dependence makes it difficult to predict the specific sites. Thus the importance of obtaining this information from direct experimental evidence is increased.

Free radical formation in X-irradiated anhydrous crystals of inosine studied by EPR and ENDOR spectroscopy.

Single crystals of anhydrous inosine were studied subsequent to exposure to high and low doses of X radiation at 10 K using K-band, EPR, ENDOR, and field-swept-ENDOR (FSE) techniques. Immediately following high radiation doses at 10 K at least eight different radicals, RI-RVIII, were observed. All radicals, except for RVIII, were also observed at low doses, but the relative yields varied with the radiation doses. RI, which decayed with no observable successor at about 65 K, has magnetic characteristics similar to those expected for the hypoxanthine base cation. RII, the dominating radical at low radiation doses, exhibits only one hyperfine coupling amenable for ENDOR analysis. From the nature of this coupling and the EPR and FSE characteristics of the resonance, it is suggested that RII is formed by addition of a neighbor sugar fragment to the C2 position of a hypoxanthine base, forming a C2-O5'-C5' ester bond. RII is unstable and decayed at about 60 K without any detectable successor. RIII and RIV are the C2 and C8 H-addition radicals, respectively. These species are formed in minor amounts after irradiation at low temperatures, and they are the only observable radicals left at room temperature. Two sugar-centered radicals, RV and RVI, are formed by net H-abstraction from the C4' and C5' positions, respectively. These radicals dominate the EPR spectra after high radiation doses at low temperatures. A transformation from RV into RIII, the C2 H-adduct, started at about 80 K. Similarly, a transformation of RVI into RIV started at about 210 K. Several minor species were analyzed. RVII is characterized by an alpha-coupling due to 26% spin density at C8, and RVIII is characterized by 12% pi-spin density at N1. Possible structures for these radicals are discussed.

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.

The structure of the guanine cation: ESR/ENDOR of cyclic guanosine monophosphate single crystals after X irradiation at 10 K.

Following X irradiation of 3',5'-cyclic guanosine monophosphate single crystals at 10 K, several free radicals were trapped and detected by ESR/ENDOR/FSE spectroscopy. The two dominant species both have unpaired spin located on the guanine base. One is the product of net hydrogen atom loss from the exocyclic amino group. The spectroscopic characteristics of this resonance leave this assignment unambiguous. The experimental conditions make it likely that this species was formed by deprotonation of the guanine base cation. The nature of the other species is more uncertain. However, the evidence is consistent with the assignment that it is a net OH adduct to the C4 position of the base. Several species in which the unpaired spin was located on the sugar-phosphate region of the molecule were also observed. The mechanisms for the decay of the primary radicals, also leading to the well-known C8 hydrogen addition radical of the guanine base, are described and discussed.

Environmental effects on primary radical formation in guanine: solid-state ESR and ENDOR of guanine hydrobromide monohydrate.

Single crystals of guanine hydrobromide monohydrate, in which the guanine base is protonated at N7, were X-irradiated at 8 and 65 K. Using K-band ESR, ENDOR, and field-swept-ENDOR (FSE) techniques, the crystals were studied between 8 K and room temperature. There was evidence for five different radicals, RI-RV, immediately following irradiation at 8 or 65 K. RI was identified as the O6-protonated anion. It decayed near room temperature with no detectable successor. RII was identified as the N7-deprotonated cation, and decayed near 130 K. RIII is thought to be a ring-opened product formed by C8-N9 bond rupture; upon warming, it decayed at 150 K. RIV is the well-known C8 H-addition radical. These four radicals have been observed previously in the hydrochloride salt of guanine monohydrate. RV is novel, however, with magnetic characteristics consistent with those of the product formed by net OH addition to C5 of the unsaturated C4-C5 bond. It is characterized by four alpha-proton couplings indicating pi-electron spin as follows: 13% at C8; 11% at N7; and 12% at N10. RV decayed between 240 and 255 K with no detectable successor. Upon further warming, very weak resonance lines due to additional, unidentified radicals were observed. A comparison of these results with those obtained from other systems containing N7-protonated guanine bases demonstrates the effect of the environment on the primary radical formation.