Radiation-induced crosslinks between thymine and phenylalanine

OH radicals generated by ionizing radiation in aqueous solutions of thymine (T) and phenylalanine (Phe) induce crosslinking between thymine and phenylalanine. The crosslinked products were isolated and characterized by capillary gas chromatography-mass spectrometry. They are formed via OH radical adducts to thymine and phenylalanine and the reaction between dissimilar radicals is greatly favoured (T-Phe:Phe-Phe:T-T = 0.46:0.14:0.05). The reaction mechanism presented may serve as a model for radiation or any free radical-induced crosslinks between DNA and proteins.     

Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

Free radical reactions with proteins and enzymes: the inactivation of pepsin.

The reactions of free radicals produced by ionizing radiation with pepsin have been studied by steady-state inactivation measurements and by pulse radiolysis. In de-aerated solutions thehydroxyl radical has been found to be the most efficient of the primary free radicals generated from water in causing inactivation. The reactions of the more selective oxidizing inorganic radical anions Br2-. and (SCN)2-., with pepsin have also beenexamined. In the case of the thiocyanate radical anion (SCN)2-., the inactivation efficiency is found to depend on SCN- concentration, an effect shown to arise from a reversible redox reaction involving the tryptophan and (SCN)2-. radicals. The results demonstrate that tryptophan residue plays an essential role in the enzyme activity of pepsin.     

Physico-Chemical Evaluation of Rationally Designed Melanins as Novel Nature-Inspired Radioprotectors

Background

Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.

Methodology/Principal Findings

We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14·10¹⁸, 7.09·10¹⁸, and 9.05·10¹⁷ spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy (¹³⁷Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.

Conclusions/Significance

We propose that due to melanin''s numerous aromatic oligomers containing multiple π-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.     

Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells is, to a large extent, due to oxidative stress. The molecule most often reported to be damaged by ionizing radiation is DNA. Hydroxyl radicals (*OH), considered the most damaging of all free radicals generated in organisms, are often responsible for DNA damage caused by ionizing radiation. Melatonin, N-acetyl-5-methoxytryptamine, is a well-known antioxidant that protects DNA, lipids, and proteins from free-radical damage. The indoleamine manifests its antioxidative properties by stimulating the activities of antioxidant enzymes and scavenging free radicals directly or indirectly. Among known antioxidants, melatonin is a highly effective scavenger of *OH. Melatonin is distributed ubiquitously in organisms and, as far as is known, in all cellular compartments, and it quickly passes through all biological membranes. The protective effects of melatonin against oxidative stress caused by ionizing radiation have been documented in in vitro and in vivo studies in different species and in in vitro experiments that used human tissues, as well as when melatonin was given to humans and then tissues collected and subjected to ionizing radiation. The radioprotective effects of melatonin against cellular damage caused by oxidative stress and its low toxicity make this molecule a potential supplement in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized.     

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.     

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M₄PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H₂O₂, which is the recombination product of ·OH, and OCl^- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.     

In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.     

Interaction between D-amino acid oxidase and small molecules.

1. From the standpoint of monomer-dimer equilibrium of hog kidney D-amino acid oxidase [EC 1.4.3.3] and the interaction between the enzyme and small molecules, the effect of pH on the binding of p-aminobenzoate to the monomer and dimer of the enzyme was studied by kinetic methods and spectrophotometric titration. 2. The maximum binding number of p-aminobenzoate to the dimer is two molecules, and there is no interaction between the two active sites of the dimer (i.e., no cooperativity) over the range of pH from 6.5 to 10. 3. The affinity of the dimer for p-aminobenzoate is several times higher than that of the monomer at pH 6.5-10, and consequently p-aminobenzoate induces dimerization in the equilibrium state of D-amino acid oxidase. The interaction energy of two subunits of the dimer is stabilized by the binding of p-aminobenzoate by 1-2 kcal/mole over the pH range studied. 4. The binding sites of the quasi-substrate, p-aminobenzoate, in the dimer and the intersubunit binding site of the dimer are clearly different, because p-aminobenzoate induces dimerization of the enzyme. 5. The pK values of ionizing groups in the free monomer and the free dimer which participate in the binding of the competitive inhibitor, p-aminobenzoate, are approximately the same, 8.7, as determined from the pH dependence of the affinity of the inhibitor for the enzyme. Furthermore, no pK for the enzyme-inhibitor complex in the pH range 6.5-10 was observed. 6. There is no interaction between the two ionizing groups of the dimer during protonation-deprotonation, because a theoretical equation involving no cooperativity between the two ionizing groups in the dimer explains the results well.     

Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol.

The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E.     

Ascorbic acid metabolism in protection against free radicals: a radiation model

The role of ascorbic acid in scavenging free radicals was evaluated in a model of mammalian colonic epithelium homogenized in physiologic buffer and exposed to ionizing radiation. Ascorbic acid interacts with hydroxyl free radicals, resulting in production of the ascorbate free radical (AFR). Colonic mucosa contains a soluble factor that is heat sensitive, PCA precipitable and is contained within 1,000 MW dialysis tubing; it uses GSH and cysteine to reduce AFR. The factor from rat colon is fractionated between 55 and 70% saturation with solid (NH4)2SO4; a 3-4 fold increase in enzyme activity was achieved. We suggest that the factor is a cytosolic enzyme appropriately referred to as soluble AFR-reductase. This information provides insight into the mechanism by which ascorbic acid protects against damage by hydroxyl free radicals.     

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.     

Yeast DNA diesterase for 3'-fragments of deoxyribose: purification and physical properties of a repair enzyme for oxidative DNA damage

The DNA strand breaks resulting from exposure to the free radicals generated by ionizing radiation or oxidizing agents are refractory to DNA repair synthesis because of deoxyribose fragments that block their 3' termini. The restoration of normal 3'-OH nucleotide primers is the essential first step in the excision repair of these radical-induced strand breaks. We have used a synthetic DNA substrate containing 3'-phosphoglycolaldehyde esters to identify and purify to physical homogeneity the major yeast diesterase that removes such nucleotide fragments. Yeast 3'-phosphoglycolaldehyde diesterase had Mr = 40,500 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular weight estimate from gel filtration indicated that the active species is a nearly globular monomer. Purification of the enzyme removed a tightly bound metal, but the activity of the purified enzyme could be restored by the addition of Co2+, Mn2+, Ni2+, or Zn2+, with Co2+ the most effective cofactor. Even 3 microM Co2+ stimulated near-maximal activity, and this metal also conferred significant thermal stability on the purified protein. This is a novel enzyme, whose N-terminal amino acid sequence does not show any significant similarity to published sequences, and which is not the product of any gene in the RAD52 epistasis group.     

Structure of a hydroxyl radical induced DNA-protein cross-link involving thymine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving thymine and tyrosine in nucleohistone is described. Hydroxyl radicals were generated in N2O-saturated aqueous solution by ionizing radiation. Samples of nucleohistone were hydrolyzed with HCl and trimethylsilylated. Analysis of irradiated samples by gas chromatography-mass spectrometry with selected-ion monitoring showed the presence of a thymine-tyrosine cross-link on the basis of typical fragment ions from the previously known mass spectrum of its trimethylsilyl derivative. The yield of this DNA-protein cross-link in nucleohistone was measured at incrementing doses of radiation and found to be a linear function of radiation dose between 14 and 300 Gy (J.kg-1). This yield amounted to 0.003 mumol.J-1. The mechanism of formation of this DNA-protein cross-link is thought to result from H atom abstraction by hydroxyl radicals from the methyl group of thymine followed by the addition of the resultant thymine radical to the carbon 3 position of the tyrosine ring and subsequent oxidation of the adduct radical.     

Global antibody development trends

Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fab^?2-to-Fab^?2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR® enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

Hydrogen peroxide-induced base damage in deoxyribonucleic acid

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.     

Specific association of bromocresol purple anions with a magnesium complex of a phosphorylated intermediate during steady-state hydrolysis of ATP by the Mg2+ + Ca2+-dependent ATPase of sarcoplasmic reticulum

The mechanism of dimeric binding of bromocresol purple (BCP) anions to Mg2+ + Ca2+-ATPase of the sarcoplasmic reticulum (SR) and the resulting partial inhibition of the ATPase activity were studied. BCP anions in three states, free monomer, bound monomer, and bound dimer, were spectrophotometrically calculated by solving simultaneous equations, delta A lambda 1-lambda 2 = sigma delta ai (epsilon i lambda 1-epsilon i lambda 2), and concentration changes of these states were analyzed. The addition of ATP caused an increase in the bound dimer and a decrease in the free monomer, but the change of the bound monomer was slight. The decrease in delta A (decrease phase) on the addition of ATP on dual-wavelength spectrophotometry at 585-610 nm was related to an increase in the amount of dimer bound to the SR membranes. The magnitude of the decrease phase increased with an increase in Mg2+ concentration and decreased with an increase in the concentration of Ca2+. BCP anions at the probe concentration partially inhibited the ATPase activity, and brought about a decrease in the ADP-sensitive E-P (E1P) and an increase in the ADP-insensitive E-P (E2P), though BCP anions did not affect the amount of total E-P. On elimination of Mg2+ at the steady-state E-P level both E2P and E2P . (BCP)2 were decomposed, suggesting that the enzyme form binding the BCP dimer was Mg . E-P. An increase in Mg2+ concentration increased E2P but an increase in Ca2+ concentration decreased E2P. Decomposition of E2P to P1 was inhibited by BCP anions. The following simple scheme was suggested to explain the partial inhibition of the ATPase activity, (Formula: see text). Application of BCP anions was discussed for use as a probe for Mg . E-P in the steady-state ATP hydrolysis.     

Target size analysis by radiation inactivation: the use of free radical scavengers

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.     

Radiation protection of Escherichia coli B/r by hydroxyl radical scavengers

We have used Escherichia coli B/r to test the proposal that hydroxyl radicals (.OH) are major contributors to lethal damage when bacteria in equilibrium with air or 100% nitrogen are exposed to ionizing radiation. In addition, we have tested the hypothesis that oxygen sensitizes bacterial cells to radiation by reacting at radical sites previously formed by reactions of .OH. Our results with B/r indicate that the involvement of OH radicals in damage may have been overestimated. We believe that simple .OH removal provides B/r with only a relatively small amount of protection in N2 and air. Although some .OH scavengers can have large protective effects in air, evidence supports the tentative conclusion that these effects are not based on simple .OH removal. If this conclusion is correct, then radiation sensitization by oxygen--at least of this bacterial strain--would be unrelated to reactions of .OH.