[en] The yields of R- and S-8,5'-cycloadenosines have been measured in poly A irradiated with gamma rays in the absence of oxygen. High performance liquid chromatographic analysis of the nucleoside analogues obtained by hydrolysis of the irradiated poly A shows that the R isomer predominates to the extent of 2.5-fold at doses of ionizing radiation in the range of 0-400 Gy

[en] Polyclonal antisera specific for 8,5'-cycloadenosine-5'-monophosphate (8,5'-cyclo-AMP) or its deoxy analog (8,5'-cyclo-dAMP) were elicited by immunizing rabbits with a conjugate prepared by the method of Johnston et al. [Biochemistry 22, 3453-3460 (1983)]. A competitive enzyme-linked immunosorbent assay (ELISA) was developed and used to detect the formation of these products in irradiated solutions of polyadenylic acid [poly(A)] or DNA which were saturated with nitrous oxide, nitrogen, or oxygen. The 8,5'-cyclo-AMP or 8,5'-cyclo-dAMP moieties could be detected in poly(A) at 1.0 krad and in native DNA at 10 krad, respectively. The yield of 8,5'-cyclo-dAMP was found to be two to three times higher in irradiated double-stranded DNA than in single-stranded DNA. The hydroxyl radical appears to initiate 8,5'-cyclonucleotide formation in irradiated nucleic acids, as demonstrated by the inhibition of 8,5'-cyclo-AMP formation in irradiated poly(A) by dimethyl sulfoxide. However, irradiation under nitrous oxide, particularly at low doses, does not lead to the expected increases in the yield of the 8,5'-cyclonucleotide

[en] The transfer of radiation damage initiated in the sugar phosphate moiety to a nucleotide base as exemplified by 8,5'-cyclonucleotide formation may be important in double-stranded nucleic acids where the bases are shielded to direct hydroxyl attack. With this in mind the authors have renewed a study of the radiation chemistry of cyclonucleotides including further development of an in situ immunochemical assay for their formation in nucleic acids. 8,5'-cycloadenosine 5'-monophosphate has been prepared by radiation chemical synthesis for this purpose. The authors have discovered that the Erlanger and Bieser technique in which the cyclonucleotide hapten is attached to bovine serum albumin (BSA) through the sugar moiety may not be the best approach for preparing cyclonucleotide-containing immunogens as conformational changes may occur in the cyclonucleotide structure during this procedure. The authors are presently using an alternate approach in which the cyclonucleotide hapten is linked to BSA through the phosphate group of the nucleotide. The authors report on these experiments as well as on the basic radiation chemistry of 8,5'-cyclonucleotides

[en] A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions

[en] The yields of 8,5'-cycloadenosine-5'-monophosphate, 8-hydroxyadenosine-5'-monophosphate, and adenine release have been measured in aqueous solutions of adenosine-5'-monophosphate (5'-AMP) irradiated under nitrous oxide. The yields are strongly pH dependent in a way which reflects the pK/sub a/'s of ionizable groups within 5'-AMP. Redistribution of hydroxyl radical attack between base and ribose moieties appears to be occurring in response to changes in the acid-base equilibria of 5'-AMP. Other pH-dependent phenomena which only indirectly reflect the pK/sub a/'s of 5'-AMP include the result that the yield of the (R) epimer of 8,5'-cycloadenosine-5'-monophosphate predominates at low pH while that of the (S) epimer predominates at high pH

[en] The transfer of radiation damage initiated in the sugar phosphate backbone to a nucleotide base as exemplified by 8,5'-cyclonucleotide formation has been investigated in polyadenylic acid, native and heat-denatured DNA. Polyclonal antiserum was raised in rabbits with a protein-8,5'-cycloadenosine-5'monophosphate (8,5'-cycloAMP) conjugate prepared by the carbodiimide method. An indirect, enzyme-linked immunosorbent assay (ELISA) was developed with this antiserum to probe for 8,5'-cycloAMP formation. The assay can readily detect product formation in polyadenylic acid irradiated to a total dose of 1.0 krad in the absence of oxygen. Product formation in native or heat-denatured DNA irradiated in 0.1 M phosphate buffer (pH 7.00) in the absence of oxygen is detected after approximately 20 krads. The authors shall extend these studies to determine the utility of immunochemical assays for investigating the radiation chemistry of nucleic acids

[en] The authors investigated DNA-protein crosslinks formed between aliphatic amino acids and thymine in calf thymus nucleohistone exposed predominantly to hydroxyl radicals in γ-irradiated N₂O-saturated aqueous solution. DNA-protein crosslinks involving thymine and the amino acids Gly, Ala, Val, Leu, Ile and Thr were identified. In some cases several isomers of the same crosslink were observed. The yield of the crosslinks was measured by GC-MS/SIM and was found to be a linear function of radiation dose in the range 49 to 436 Gy. The mechanism for the formation of these DNA-protein crosslinks is thought to involve hydrogen atom abstraction by hydroxyl radicals from the aliphatic amino acid followed by addition of the amino acid radical to the carbon(6)-position of thymine and subsequent oxidation of the adduct radical. (author)

[en] Solvated electrons generated in aqueous solution after exposure to ionizing radiation can be scavenged by DNA and then transferred along the DNA molecule. This mechanism of charge transfer provides an opportunity for radiation damage to be targeted to certain regions in the DNA molecule and is a mechanism by which single-strand breaks contribute to locally multiply damaged sites to enhance cell lethality. Experiments were performed in which different amounts of 5-bromouracil (5-BrU) were substituted for thymine in Escherichia coli DNA. The amount of bromide released was assayed after quantitative reaction of radiation-induced solvated electrons with 5-BrU in DNA samples irradiated in solution and irradiated in the cellular environment. By varying the amount of 5-BrU incorporated in the DNA, the average distance between 5-BrU molecules was systematically changed and, because the number of 5-BrU/electron reactions was monitored by the amount of bromine released, the maximum average electron migration distance along the 5-BrU DNA could be estimated. Using this approach, the maximum average electron migration distance in aqueous solutions of 5-BrU DNA was about 6.5 to 10 base distances in nonhybrid 5-BrU DNA (assuming only intrastrand migration). Similar methods revealed charge migration in 5-BrU DNA incorporated into E. coli, and the maximum average migration distance was about 5 to 6 base distances (assuming only intrastrand migration). Only 11-16% of the electrons produced during radiolysis were scavenged by 5-BrU DNA in aqueous solution, and only 1% resulted in the release of bromide from 5-BrU-DNA inside E. coli. 40 refs., 5 figs

[en] Radiolytic depurination, a major process in irradiated adenine nucleotides, can be distinguished from other types of damage such as 8-hydroxyadenine and 8,5'-cycloadenosine formation in the following ways. It is less sensitive to the presence of molecular oxygen than 8-hydroxyadenine formation. Depurination in 5'-mononucleotides is insensitive to pH changes in the range 4 - 10 whereas the yields of both other products show strong dependencies over this range. Depurination can also be distinguished kinetically in that it appears to be less sensitive to the inhibiting effects of dimethylsulfoxide (DMSO) than the other two processes. The details of the radical reactions underlying these differences between depurination and other types of damage may have a bearing on our understanding of the cellular repair of nucleic acid damage

[en] The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity. (author)