International Journal of Radiation Biology, June 2014; 90(6): 416–422 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.908040
WEISS LECTURE †
Carbohydrate radicals: from ethylene glycol to DNA strand breakage* Clemens von Sonntag
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Max-Planck-Institut für Strahlenchemie, Stiftstr. 34–36, Mülheim/Ruhr, F.R. Germany It was in the late forties that Josef Weiss, Gabriel Stein and George Scholes started to investigate the radiation chemistry of nucleic acids (Scholes et al. 1949). It was pioneering work indeed because even the solvated electron was not yet fully established as an important species in the radiolysis of water. Nucleic acids were by no means commercially available and the knowledge of the chemistry of their constituents was limited, not to speak of their free-radical chemistry. A similar situation prevailed in the field of carbohydrate radiation chemistry where the first steps were soon afterwards to be made by both G. O. Phillips and N. K. Kochetkov (for reviews see Phillips 1961, 1971, Kochetkov et al. 1979, von Sonntag 1980). It was not before the late sixties that we became interested in the carbohydrate field as well, actually not so much in its own right but rather with an eye on the nucleic acids as our guiding star, where the sugar moiety must play an important role in the radiation-induced strand breaking process. This phenomenon was discovered by Taylor et al. (1948) and studied in some detail by Hagen (1964, 1967). Having had no experience in carbohydrate chemistry, it very soon became obvious to us that we would not be able to make any progress in this field if we started out with such relatively complex molecules, the analytics of which were not yet sufficiently well established. We therefore went back one further step and chose as the primary target of our investigation ethylene glycol, which represents one essential feature of carbohydrates, namely the vicinal glycol structure. From the product study (Seidler and von Sonntag 1969, von Sonntag and Thorns 1970) it soon became clear that the governing step of its free radical chemistry is a water elimination reaction (reaction (1)).
Abstract Radiation-induced DNA strand breakage results from the reactions of radicals formed at the sugar moiety of DNA. In order to elucidate the mechanism of this reaction investigations were first performed on low molecular weight model systems. Results from studies on deoxygenated aqueous solutions of ethylene glycol, 2-deoxy-D-ribose and other carbohydrates and, more relevantly, of D-ribose-5-phosphate have shown that substituents can be eliminated from the β-position of the radical site either proton and base-assisted (as in the case of the OH substituent), or spontaneously (as in the case of the phosphate substituent). In DNA the C(4’) radical undergoes strand breakage via this type of reaction. In the presence of oxygen the carbon-centred radicals are rapidly converted into the corresponding peroxyl radicals. Again, low molecular weights models have been investigated to elucidate the key reactions. A typical reaction of DNA peroxyl radicals is the fragmentation of the C(4’)-C(S’) bond, a reaction not observed in the absence of oxygen. Although OH radicals may be the important direct precursors of the sugar radicals of DNA, results obtained with poly(U) indicate that base radicals may well be of even greater importance. The base radicals, formed by addition of the water radicals (H and OH) to the bases would in their turn attack the sugar moiety to produce sugar radicals which then give rise to strand breakage and base release. For a better understanding of strand break formation it is therefore necessary to investigate in more detail the reactions of the base radicals. For a start, the radiolysis of uracil in oxygenated solutions has been reinvestigated, and it has been shown that the major peroxyl radical in this system undergoes base-catalysed elimination of O-2• , a reaction that involves the proton at N(l). In the nucleic acids the pyrimidines are bound at N(l) to the sugar moiety and this type of reaction can no longer occur. Therefore, with respect to the nucleic acids, pyrimidines are good models only in acid solutions where the O-2• elimination reaction is too slow to compete with the bimolecular reactions of the peroxyl radicals. Moreover, the long lifetime of the radical sites on the nucleic acid strand may allow reactions to occur which are kinetically of first order, and which cannot be studied in model systems at ordinary dose rates. It is therefore suggested to extend model system studies to low dose rates and to oligonucleo-tides. Such studies might eventually reveal the key reactions in radical-induced DNA degradation.
CHOH CH2OH → H2O CHO CH2
(1)
This interpretation was aided by e.s.r. studies where the process was. directly observed, at least at low pH (Bazhin et al. 1966, Buley et al. 1966, Livingston and Zeldes 1966). The water elimination reaction is both base- and acid-catalysed (Bansal et al. 1973) but at the low dose rate of 60Co γ-radiolysis it is fast enough to proceed to the extent that it governs the product distribution even in neutral solutions. The products shown in Table I are explained by the disproportionation and dimerization of the two radicals
†Given on the occasion of the presentation at the Association for Radiation Research meeting in Dublin, April 1984, of Weiss medals to Professors D. Schulte-Frohlinde and C. von Sonntag for their work on the radiation chemistry of DNA and its components. *This is a re-publication of an article published in a past International Journal of Radiation Biology issue (Von Sonntag, Clemens (1984) ‘Weiss Lecture’, International Journal of Radiation Biology, 46:5,507–519) for this special issue in honour of Professor Clemens von Sonntag.
(Received 12 June 1984)
416
Carbohydrate radicals 417
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Table I. γ-Radiolysis of ethylene glycol (101 m) in N2O-saturated aqueous solutions at various temperatures and a dose rate of ≃ 0.1 Gy s-1. Products and their G values according to von Sonntag and Thorns (1970). G value Products
0°C
20°C
80°C
Succindialdehyde Glycolaldehyde 2-Deoxytetrose Tetritol Acetaldehyde
1.9 0.9 0.3 0.15 0.5
1.7 1.1 0.25 0.15 1.2
2.3 0.8 0.13 0.03 6.4
shown in reaction (1). Also, favoured by elevated temperatures, low dose rate and high ethylene glycol concentration, acetaldehyde is formed via a chain reaction, the propagating step being reaction (2). CH2 CHO CH2OH CH2OH → CH3 CHO CHOH (2) CH2OH Analytical techniques were then refined (Dizdaroglu et al. 1972, 1974, 1977b) and it was eventually possible to fully analyse products from complex molecules such as 2-deoxyd-ribose (Hartmann et al. 1970) or d-glucose (cf. Table II), Table II. Products and their G values from the γ-radiolysis of deoxygenated, N2O-saturated or 4:1 N2O/O2-saturated aqueous solutions of d-glucose atadose rate of 0.18 Gy s1 at room temperature according to Dizdaroglu et al. (1975a) and Schuchmann and von Sonntag (1977). G value Product
N 2O
N2O/O2
d-Gluconic acid d-arabino-Hexos-2-ulose d-ribo-Hexos-3-ulose d-xylo-Hexos-4-ulose d-xylo-Hexos-S-ulose d-gluco-Hexodialdose 2-Deoxy-d-arabino-hexonic acid 5-Deoxy-d-threo-hexos-4-ulose 5-Deoxy-d-xylo-hexonic acid 2-Deoxy-d-erythro-hexos-5-ulose 5-Deoxy-d-xylo-hexodialdose 3-Deoxy-d-erythro-hexos-4-ulose 3-Deoxy-d-erythro-hexos-2-ulose 4-Deoxy-l-threo-hexos-5-ulose 6-Deoxy-d-xylo-hexos- 5 -ulose 2-Deoxy-d-erythro-hexos-3-ulose 4-Deoxy-d-threo-hexos-3-ulose d-Arabinose d-Arabinonic acid d-Ribose d-Xylose xylo-Pentodialdose 2-Deoxy-d-erythro-pentose d-Erythrose d-Erythronic acid l-Threose l-threo-Tetrodialdose 3-Deoxytetrulose 1,3-Dihydroxy-2-propanone d-Glyceraldehyde and glyceric acid Glyoxal Glyoxylic acid/glycolic acid Formaldehyde Formic acid d-Glucose consumption
0.15 0.15 0.10 0.075 0.18 0.22 0.95
0.90 0.90 0.57 0.50 0.60 1.55 absent absent absent absent absent absent absent absent absent absent absent 0.10
}
}
† Products identified (no
0.08
0.25 0.05 † † 0.01 absent 0.005 0.005 absent 0.04 0.01 absent 0.003 absent 0.02 0.03 absent ‡ ‡ ‡ ‡ 5.6
}
except for the dimer fraction, which in the case of deoxygenated solutions of glucose must contain more than a hundred different products. The discovery of the water elimination reaction, the knowledge of the nature of the products from this reaction, and the necessary analytics were timely, because soon it was recognized that this type of reaction is not restricted to the elimination of water, but, much more important for DNA research, that a similar reaction also prevails in phosphate esters, as shown by e.s.r. using glycerol phosphates as substrates (Samuni and Neta 1973, Steenken et al. 1974) and by product studies on d-ribose-5-phosphate (Stelter et al. 1974, 1975, 1976) as well as d-fructose phosphates (Zegota and von Sonntag 1981). Mechanistically, the phosphate elimination is somewhat different from the water elimination as it is already quite fast in neutral solutions, i.e. the reaction does not need to be speeded up by protons or hydroxide ions in order to become detectable by e.s.r. spectroscopy. In 1975 the first altered sugars resulting from radiationinduced DNA strand breakage were identified (Dizdaroglu et al. 1975 b). It had been postulated that radical cations might play a role as intermediates (cf. reaction (3)). Although at the time this was only a working hypothesis and other alternative mechanisms had to be considered (cf. von Sonntag et al. 1981) it was later shown that radical cations are the most likely intermediates (Behrens et al. 1978a, 1982); in special cases such species are sufficiently stable against water attack so that their e.s.r. spectra can be recorded (Behrens et al. 1978 b).
absent 0.08
}
absent 0.02 absent 0.20 absent absent 0.13 0.11 0.4 0.12 0.6 5.6
G values given). They are expected to be included in the G values of the other deoxyhexosuloses given in the table. ‡ Not determined, probably absent.
Armed with the knowledge of the principles of the carbohydrate free radical reactions—as the result of detailed studies of some further small model systems—we realized that
418
C. von Sonntag
sugars similar to those which were set free from the DNA on irradiation must also remain bound to DNA by the phosphate linkage. Hence techniques were developed to excise such lesions from irradiated DNA (Dizdaroglu et al. 1977a, Beesk et al. 1979). The altered sugars recognized so far, both free and bound to DNA, are shown in Table III. Not all of these alterations are linked with immediate strand breakage. Rather products 1, 2 and 12, and possibly 3, represent alkali-labile sites which turn into strand breaks upon alkali
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Table III. Alterations at the sugar moiety in the γ-radiolysis of DNA in deoxygenated and oxygenated solutions and site of precursor radicals. For references see text.
treatment. As shown in table III, some of the products appear only if the irradiation is carried out in the presence of oxygen. Besides these lesions which have been discovered at Mülheim, another lesion, brought about in the presence of oxygen, has been repeatedly reported, which is characterized by giving a positive 2-thiobarbituric acid (TBA) test (Krushinskaya and Shalnov 1967, Kapp and Smith 1970, Ullrich and Hagen 1971). The formation of glycolic acid bound to DNA 9 has also been reported (Henner et al. 1983). These observations would be compatible if in DNA a lesion of the type 13 was formed, similar to the products that have been suggested to be prominently related to bleomycin-induced strand breakage (Burger et al. 1980, Giloni et al. 1981). In the radiolytic system this TBA-active lesion still calls for a detailed analysis.
Because of the omnipresence of oxygen in nature and the sensitizing effect that oxygen exhibits in cellular systems, we became strongly interested in the effect of oxygen on these systems. Again, it had been J. Weiss who pioneered this field; he had also realised that one would have to investigate low molecular weight systems to obtain some insight into the general features. Being the modest and careful scientist he was, he considered it premature at the time of his investigations of the methane (Johnson and Weiss 1955) and ethylene systems (Clay et al. 1958) to put forward a mechanistic proposal to explain the products which he had observed. Since then, we have learned much about peroxyl radicals, and nowadays pulse radiolysis and more sophisticated analytical techniques allow us to reinvestigate these systems in more detail (Schuchmann and von Sonntag 1984, Piesiak et al. 1984) and to make mechanistic proposals. Reactions (13)—(22) represents our present knowledge of the decay of primary and secondary peroxyl radicals.
With a few exceptions (see below), peroxyl radicals decay bimolecularly. In the first step a short-lived tetroxide is formed (reaction (13)) which at low temperatures is in equilibrium with the peroxyl radicals (cf. reaction (14)) (Bennett et al. 1970) but which at elevated temperatures readily decomposes into products (Furimsky et al. 1980). Three concerted
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Carbohydrate radicals 419 processes have been recognized so far (reactions (15)–(17)), and one leading to molecular oxygen and two oxyl radicals (reaction (18)). The best known concerted process is the so-called Russell mechanism which leads to 02, alcohol and a carbonyl compound (reaction (15)) via a six-membered transition state (Russell 1957, Howard and Ingold 1968). The transition state of the second concerted process, the formation of H202 and two carbonyl compounds (reaction (16)) is likely to involve two five-membered rings (Bennett and Summers 1974, Bothe and Schulte-Frohlinde 1978, Schuchmann and von Sonntag 1979). It has been recently shown (Zegota et al. 1984, Piesiak et al. 1984) that the welldocumented (Howard 1973) fragmentation of the carboncarbon skeleton is also very likely to be due to a concerted process (reaction (17)). This reaction had earlier been postulated to involve free oxyl radicals (reaction (22)). Oxyl radicals carrying an a-hydrogen rearrange in water so quickly into the corresponding α-hydroxyalkyl radicals (reaction (19)) (Berdnikov et al. 1972, Gilbert et al. 1976, 1977, Schuchmann and von Sonntag 1981) that the fragmentation reaction (22) cannot compete effectively. The combination of two peroxyl radicals (reaction (20)) is usually of minor importance. Product-wise, the disproportionation reaction (21) cannot be distinguished from the Russell mechanism (reaction (15)). The α-hydroxyalkyl peroxyl radical formed by O2 addition to the α-hydroxyalkyl radicals can eliminate HO2 or, baseinduced, O-2• (reactions (23) and (24)).
Such radicals are not only formed in the course of the bimolecular decay of peroxyl radicals (Schuchmann and von Sonntag 1982) but more directly by OH-attack on alcohols and carbohydrates (Rabani et al. 1974, Ilan et al. 1976, Bothe et al. 1977, 1978b, 1983, for a review see Bothe et al. 1978a). For example, the major products observed in the radiolysis of d-glucose in oxygenated solutions (cf. Table II) are formed according to such a reaction (Schuchmann and von Sonntag 1977). In DNA there are no structural elements which, after OH attack and oxygen addition, would directly lead to the formation of α-hydroxyalkylperoxyl radicals. However, such radicals can also be formed by the sequence of the reactions (14)-(19). In competition there is almost always the fragmentation reaction (22). This type of reaction (or its concerted equivalent, reaction (17)) also appears to play a role in the formation of DNA lesions 2, 9, 10 and 11 (cf. Table III). With the exception of lesion 12 in all the altered sugars that have been observed in DNA so far (cf. Table III) an unaltered base is lost as well. This has led to an investigation of base release in a model system, poly(U) (Deeble and von Sonntag 1984) (Table IV). With this system it has been clearly shown that the release of unaltered base is induced by another base radical corroborating other evidence obtained in our institute (Lemaire et al. 1984) that base radicals induce strand breakage. The same reasoning put forward for deoxygenated systems can be extended to oxygenated ones
Table IV. Products and their G values in the g-radiolysis of N20 and N20/02 (4:1 )-saturated solutions of poly(U). G values N2O Strand breakage Base release (C) Oxygen uptake (d) Organic (hydro)peroxide (e) Hydrogen peroxide (e) Carbon dioxide (e) Osazone-forming compounds (e) Organic acid (pulse radiolysis) (f )
N2O/O2
2.3 (a) 2.9 — — n.d. absent 0.1 —
3.5 (b) 3.5 20 7 1.2 2.2 2.7 2.0
References: (a) Lemaire et al. (1984), (b) Lemaire cited by Schulte-Frohlinde and Bothe (1984), (c) Deeble and von Sonntag (1984), (d) Isildar et al. (1982), (e) Deeble and von Sonntag, unpublished results, (f ) Schulte-Frohlinde and Bothe (1984).
(Schulte-Frohlinde and Bothe 1984). If such a process were also to occur in DNA the sugar radicals ultimately responsible for strand breakage may well have base radicals as precursors. This brings us back to the free-radical chemistry of nucleobases, one of the early main research topics of Jo Weiss. Our group joined the research effort in this field some time ago (Campbell et al. 1974a, Hissung and von Sonntag 1978, 1979, Hissung et al. 1981, Schuchmann and von Sonntag 1983, Al-Sheikhly and von Sonntag 1983, Schuchmann et al. 1984b), and we have again experienced the old Newcastle stimulus in the cooperation with his old friend George Scholes (cf. Al-Sheikhly et al. 1984, Schuchmann et al. 1984a). In particular, the reinvestigation (for earlier work see Latarjet et al. 1962, Smith and Hays 1968, Peuzin et al. 1970, Ducolomb et al. 1971, 1973, Giroud et al. 1980) of the radiolysis of uracil in oxygenated solutions (Schuchmann et al. 1984b) has yielded interesting new information. The main (cf. Fujita and Steenken 1981) peroxyl radical in this system can undergo a base-induced O-2• -elimination (reaction (25)) which leads to isobarbituric acid and the glycols (reactions (26) and (27)) (Table V). In competition there is a bimolecular decay of which the typical products are isodialuric acid and formylhydroxyhydantoin (reactions (28)-(34)). Whereas at high pH, and also in neutral solutions at low dose rates, the O-2• -elimination reaction predominates, the bimolecular decay becomes prominent at low pH and in neutral solutions at high dose rates. Thus the simple model uracil is quite a complex system. The O-2• -elimination involves the proton at N(l). In the nucleic acids this proton is replaced by the sugar moiety, hence O-2• -elimination no longer occurs. Thus uracil irradiated in neutral solutions at low dose rates is not a perfect model Table V. γ-Radiolysis of N2O/O2-saturated uracil (2 104M) solutions under various pH conditions. G value Product 5,6-cis-Dihydroxy-5,6-dihydrouracil 5,6-trans-Dihydroxy-5,6-dihydrouracil Isobarbituric acid 1-N-Formyl-5-hydroxy-hydantoin Dialuric acid Isodialuric acid 5-Hydroxyhydantoin Unidentified product(s) Hydrogen peroxide Oxygen consumption Uracil consumption
pH3-0
pH6-5
pH100
0.6 0.5 0 1.6 0.9 0.1 0.4 0.9 n.d. n.d. 4.9
0.9 1.1 0.2 1.4 0.4 0.2 0.4 0.6 3.0 5.0 5.3
1.4 1.0 1.2 0.2 0.2 0.1 0.3 0.9 n.d. n.d. 5.2
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
420
C. von Sonntag
to study the fate of the pyrimidine peroxyl radical in nucleic acids. At first sight the uracil system studied in acid medium appears to be a better choice. Although this might well be so, one must always consider that in nucleic acids the radicals have very long lifetimes. Thus they can undergo reactions kinetically of (pseudo)first order which are not observed at the usual dose rates. We therefore suggest that model systems, e.g. small oligonucleotides, should be studied at very low dose rates. Such studies might reveal new reaction pathways hitherto not observed. In fact, base release from a nucleoside shows a pronounced dose rate effect at low pH (Fujita 1984). I like to think that Jo Weiss would agree wholeheartedly that we must continue to investigate model systems, that in fact it is impossible to sidestep the model systems, in order to arrive at a better understanding of the subject that we are primarily interested in: the radiation-induced changes of DNA within the cell.
References Al-Sheikhly M, Garner A , Hissung A , Schuchmann MN, Schuchmann, H.-P., Scholes, G., and von Sonntag, C. 1984. Radiolysis of dihydrouracil and dihydro-thymine in aqueous solutions contining oxygen; first- and second-order reactions of the rganic peroxyl radicals; the role of isopyrimidines as intermediates. J. chem. Soc. Perkin Trans. 2:601–8.
Al-Sheikhly M., and von Sonntag. C . 1983. γ-Radiolysis of 1,3-dimethyluracil in N2O-saturated aqueous solutions. Z. Naturf. b, 38:1622–9. Bansal KM, Grätzel M, Henglein A , and Janata E. 1973. Polarographic and optical absorption studies of radicals in the pulse radiolysis of aqueous solutions of ethylene glycol. J. phys. Chem., 77:16–19. Bazhin NM, Kuznetsov EV, Bubnov NN, and Voevodskii VV. 1966. Reaction of a hydrogen atom in the system H2O H2SO4 FeSO4. III. Reaction with saturated organic compounds, Kin. Katal. USSR (translated version) 7:643–5. Beesk F, Dizdaroglu M, Schulte-Frohlinde D, and von Sonntag C. 1979. Radiation-induced DNA strand breaks in deoxygenated aqueous solution. The formation of altered sugars as end groups. Int. J. Radiat. BioL 36:565–76. Behrens G, Bothe E, Eibenberger J, Koltzenburg G, and Schulte-Frohlinde D. 1978b. Nachweis eines Dialkoxyalken-Radikalkations in wässriger Lösung. Angew. Chem 90:639. Behrens G, Koltzenburg G, Ritter A , and Schulte-Frohlinde D. 1978a, The influence of protonation or alkylation of the phosphate group on the e.s.r. spectra and on the rate of phosphate elimination from 2-methoxyethyl phosphate 2-yl radicals. Int. J. Radiat. Biol 33:163–71. Behrens G, Koltzenburg G, and Schulte-Frohlinde D. 1982, Model reactions for the degradation of DNA-4’ radicals in aqueous solution. Fast hydrolysis of α-alkoxyalkyl radicals with a leaving group in β position followed by radical rearrangement and elimination reactions. Z. Naturf. c, 37:1205–27. Bennett JE, Brown DM, and Mile B. 1970. Studies by electron spin resonance of thereactions of alkylperoxy radicals. Part 2. Equilibrium between alkylperoxy radicals and tetroxide molecules. Trans. Faraday Soc 66:397–405.
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Carbohydrate radicals 421 Bennett JE, and Summers R. 1974. Product studies of the mutual termination reactions of sec-alkylperoxy radicals: Evidence for non-cyclic termination. Can. J. Chem 52:377–9. Berdnikov VM, Bazhin NM, Fedorov VK, and Polyakov OV. 1972. Isomerization of the ethoxyl radical in aqueous solution. Kinet. Katal 13:86–7. Bothe E, Behrens G, and Schulte-Frohlinde D. 1977, Mechanism of the first order decay of 2-hydroxypropyl-2-peroxyl radicals and of O2• formation in aqueous solution. Z. Naturf. b, 32:86–9. Bothe E, Schuchmann MN, Schulte-Frohlinde D. and von Sonntag C. 1978a. HO2 elimination from O-hydroxyalkylperoxyl radicals in aqueous solution. Photochem. Photobiol 28:639–44. Bothe E, Schuchmann MN, Schulte-Frohlinde D. and von Sonntag C. 1983.Hydroxyl radical-induced oxidation of ethanol in oxygenated aqueous solutions. A pulse radiolysis and product study. Z. Naturf. b, 38:12–19. Bothe E, and Schulte-Frohlinde D. 1978. The bimolecular decay of the α-hydroxymethylperoxyl radical in aqueous solution. Z. Naturf. b, 33:786–8. Bothe E, Schulte-Frohlinde D, and von Sonntag C. 1978b. Radiation chemistry of carbohydrates. Part 16. Kinetics of HO2 elimination from peroxyl radicals derived from glucose and polyhydric alcohols. J. chem. Soc. Perkins Trans 2:416–20. Buley AL, Norman R.O.C, and Pritchett RJ. 1966. Electron spin resonance studies of oxidation. Part VIII. Elimination reactions of some hydroxyalkyl radicals. J. chem Soc. (B). 849-52. Burger RM, Berkowitz AR, Peisach J, and Horwitz SB. 1980. Origin of malondialdehyde from DNA degraded by Fe{II}. bleomycin. J. biol. Chem 255:11832–8. Campbell JM, Schulte-Frohlinde D, and von Sonntag C. 1974a. Quantum yields in the u.v. photolysis of 5-bromouracil in the presence of hydrogen donors. Photochem. Photobiol 20:465–7. Campbell JM, von Sonntag C, and Schulte-Frohlinde D. 1974b, Photolysis of 5-bromouracil and some related compounds in solution. Z. Naturf. b, 29:750–7. Clay PG, Johnson G.R.A , and Weiss J. 1958, Chemical action of ionising radiations in solution. Part XXI. The action of 60-Co-gammaradiation on aqueous solutions of ethylene at different pressures. J. chem. Soc 2175–82. Deeble DJ, and von Sonntag C. 1984. γ-Radiolysis of poly(U) in aqueous solution. The role of primary sugar and base radicals in the release of undamaged uracil from poly(U). Int. J. Radiat. Biol 46:247–260. Dizdaroglu M, Henneberg D, Schomburg G, and von Sonntag C. 1975a. Radiation chemistry of carbohydrates. VI. γ-Radiolysis of glucose in deoxygenated N2O-saturated aqueous solution. Z. Naturf. b, 30, 416–25. Dizdaroglu M, Henneberg D, and von Sonntag C. 1974. The mass spectra of TMS-ethers of deuterated polyalcohols. A contribution to the structural investigation of sugars. Org. Mass Spectrom 8:335–45. Dizdaroglu M, Henneberg D, von Sonntag C. and Schuchmann MN. 1977b. Mass spectra of trimethylsilyl di-O-methyloximes of adosuloses and dialdoses. Org. Mass Spectrom 12:772–5. Dizdaroglu M, Scherz H, and von Sonntag C. 1972. Strahlenchemie von Alkoholen-XVI. γ-Radiolyse von meso-Erythrit in wassriger Losung. Z. Naturf. b, 27:29–41. Dizdaroglu M, Schulte-Frohlinde D, and von Sonntag C. 1977a. γ-Radiolysis of DNA in oxygenated aqueous solution. Structure of an alkali-labile site. Z. Naturf, c, 32:1021–2. Dizdaroglu M, von Sonntag C, and Schulte-Frohlinde D. 1975b. Strand breaks and sugar release by γ-irradiation of DNA in aqueous solution. J. Am. Chem. Soc 97:2277–8. Ducolomb R, Cadet J, and Teoule R. 1971. Effect des rayon y sur l’uracil et l’acide uridylique en solution aqueuse aeree. C. R. Acad. Sci. Paris, Ser. D., 273, 2647–9; 1973, Effet du rayonnement gamma sur l’uracile en solution aqueuse aeree. Bull. Soc. Chim. France 1167–74. Fujita S, 1984. Radiolysis of nucleosides in aqueous solutions: base liberation by the baseattack mechanism. Int. J. Radiat. Biol 45: 371–7. Fujita S, and Steenken S. 1981. Pattern of OH radical addition to uracil and methyl-and carboxyl-substituted uracils. Electron transfer of OH adducts with N,N, N’ N’-tetramethyl-p-phenylenediamine and tetranitromethane. J. Am. Chem. Soc 103:2540–5. Furimsky E, Howard JA , and Selwyn J. 1980. Absolute rate constants for hydrocarbon autoxidation. 28. A low temperature kinetic electron spin resonance study of the self-reactions of isopropylperoxy and related alkylperoxyl radicals in solution. Can. J. Chem. 58:677–80. Gilbert BC, Holmes RGG, Lue, HAH. and Norman ROC. 1976. Electron spin resonance studies. Part L. Reactions of alkoxyl radicals
generated from alkyl-hydroperoxides and titanium (III) ion in aqueous solution. J. chem. Soc Perkins Trans. 2:1047–52. Gilbert BC, Holmes RGG, and Norman R.O.C. 1977. Electron spin resonance studies. Part LII. Reactions of secondary alkoxyl radicals. J. chem. Res. (S), 1. Giloni L, Takeshita M, Johnson F, Iden C, and Grollman AP. 1981. Bleomycin-induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J. biol. Chem. 256:8608–15. Giroud AM, Cadet J, and Ducolomb R. 1980. Etude par resonance magnetique nucleaire du carbone-13 des derives de degradation radio-induite de la thymine et del’uracile. N. J. Chim. 4:657–61. Hagen U. 1964. Untersuchungen über die Strahlenempfindlichkeit der Desoxyribo-nukleinsäure. II. Die Länge der Nukleotidketten nach Bestrahlung in vitro. Strahlen-therapie, 124:428–37;1967, Bestimmung von Einzel- und Doppelbrüchen in be-strahlter Desoxyribonucleinsäre durch die Molekulargewichtsverteilung. Biochim. biophys. Acta. 134:45–58. Hartmann V, von Sonntag C, and Schulte-Frohlinde D. 1970. γ-Radiolyse von 2-Desoxyribose in wässriger Lösung. Z. Naturf. 6, 25:1394–04. Henner WD, Rodriguez LO, Hecht SM, and Haseltine WA . 1983. Gamma-ray induced deoxyribonucleic acid strand breaks: 3’glycolate termini. J. biol. Chem. 258:711–13. Hissung A , and von Sonntag C. 1978, Radiolysis of cytosine, 5-methylcytosine and 2-deoxycytosine in deoxygenated aqueous solution. A pulse spectroscopic and pulse conductometric study of the OH adduct. Z. Naturf. b, 33, 321–8; 1979, The reaction of solvated electrons with cytosine, 5-methylcytosine and 2′-deoxycytidine in aqueous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study. Int. J. Radiat. Biol 35:449–58. Hissung A, von Sonntag C, Veltwisch D, and Asmus K.-D. 1981. The reaction of the 2’-deoxyadenosine electron adduct in aqueous solution. The effects of the radiosensitizer p-nitroacetophenone. A pulse spectroscopic and pulse conductrometric study. Int. J. Radiat. Biol. 39:63–71. Howard JA . 1973. Homogeneous liquid-phase autoxidations. Free Radicals, edited by J. K. Kochi, Vol. 2 (New York: Wiley), pp. 3–62. Howard JA , and Ingold KU. 1968. The self-reaction of sec-butylperoxy radicals. Confirmation of the Russell mechanism. J. Am. chem. Soc. 90:1056–8. Ilan Y, Rabani J, and Henglein A . 1976. Pulse radiolytic investigations of peroxy radicals produced from 2-proponal and methanol. J. phys. Chem. 80:1558–65. Isildar M, Schuchmann MN, Schulte-Frohlinde D, and von Sonntag C. 1982. Oxygen uptake in the radiolysis of aqueous solutions of nucleic acids and their constituents. Int. J. Radiat. Biol, 41: 525–33. Johnson GRA , and Weiss J. 1955. The formation of methyl hydroperoxyde from methane irradiated by X-rays (200 kV) in aqueous solution in the presence of dissolved oxygen. Chem. Ind, 358-9. Kapp DS, and Smith KC. 1970. Chemical nature of chain breaks in DNA by X-irradiation in vitro. Radiat. Res. 42:34–49. Kochetkov NK, Kudrjashov LI, and Chlenov MA . 1979. Radiation Chemistry of Carbohydrates, translated by G. O. Phillips (Oxford: Pergamon Press). Krushinskaya NP, and Shalnov MI. 1967. The nature of breaks of the DNA chain by irradiation of its water solutions. Radiobiology 7:36–45. Latarjet R, Ekert B, Apelgot S, and Rebeyrotte N. 1962. Etudes radiobiochimiques sur l’ADN. J. chim. Phys., 58:1046–57. Lemaire DGE, Bothe E, and Schulte-Frohlinde D. 1984. Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals. Int. J. Radiat. Biol 45:351–8. Livingstone R, and Zeldes H. 1966. Paramagnetic resonence study of liquids during photolysis. III. Aqueous solutions of alcohols with hydrogen peroxide. J. Am. chem. Soc 88:4333–6. Peuzin MC, Cadet J, Polverelli M, and Teoule R. 1970. Parabanic acid and 5-hydroxy hydantoin upon y-irradiation of aerated aqueous solutions of uracil. Biochim. biophys. Acta 209:573–4. Phillips GO, 1961. Radiation chemistry of carbohydrates. Adv. Carbohydr. Chem. Biochem., 16, 13–58; 1971, Effects of ionizing radiations on carbohydrate systems. Radiat. Res. Rev 3:335–51. Piesiak A , Schuchmann MN, Zegota H. and von Sonntag C. 1984. (β-Hydroxyethylperoxyl radicals: A study of the y-radiolysis and pulse radiolysis of ethylene in oxygenated aqueous solutions. Z. Naturf. b, 39, (in the press). Radani J, Klug-Roth D, and Hengi.ein A . 1974, Pulse radiolytic investigations of OHCH2O2 radicals. J. phys. Chem 78:2089–93.
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
422
C. von Sonntag
Russell GA , 1957, Deuterium-isotope effects in the autoxidation of aralkyl hydrocarbons. Mechanism of the interaction of peroxy radicals. J. Am. chem. Soc 79:3871–7. Samuni A , and Neta P. 1973, Hydroxyl radical reactions with phosphate esters and the mechanism of phosphate cleavage. J. phys. Chem 77:2425–9. Scholes G, Stein G, and Weiss J. 1949, Action of X-rays on nucleic acids. Nature, Lond 164:709–10. Schuchmann H.-P, and von Sonntag C. 1981. Photolysis at 185 nm of dimethyl ether in aqueous solution: involvement of the hydroxymethyl radical. J. Photochem 16:289–95;1984, Methylperoxyl radicals: a study of the γ-radiolysis of methane in oxygenated solutions. Z. Naturf. b, 39:217–21. Schuchmann MN, Al-Sheikhly M, von Sonntag C, Garner A. and Scholes G. 1984a. The kinetics of the rearrangement of some isopyrimidines to pyrimidines studied by pulse radiolysis. J. chem. Soc. Perkin Trans. 2 (in the press). Schuchmann MN, and von Sonntag C. 1977. Radiation chemistry of carbohydrates. Part 14. Hydroxyl radical-induced oxidation of d-glucose in oxygenated aqueous solution. J. chem. Soc. Perkin Trans 2:1958-63; 1979, Hydroxyl radical-induced oxidation of 2-methyl-2propanol in oxygenated aqueous solution. A product and pulse radiolysis study. J. phys. Chem 83:780-4;1982, Hydroxyl radical induced oxidation of diethylether in oxygenated aqueous solution. A product and pulse radiolysis study. J. phys. Chem 86,1995–2000;1983, The radiolysis of uracil in aqueous solutions. A study by product analysis and pulse radiolysis. J. chem. Soc. Perkin Trans 2:1525–31. Schuchmann MN, Steenken S, Wroblewski J, and von Sonntag C. 1984b. Site of OH attack on dihydrouracil and some of its methyl derivatives. Int. J. Radiat. Biol 46:225–232. Schulte-Frohlinde D, and Bothe E. 1984. Identification of a major pathway of strandformation in poly U induced by OH radicals in presence of oxygen. Z. Naturf. c, 39:315–19. Seidler F, and von Sonntag C. 1969. Strahlenchemie von Alkoholen– VIII. Die Acetaldehydbildung bei der γ-Radiolyse wässriger, N2Ogesättigter L ösungen von Äthylenglykol. Z. Naturf. b, 24:780–1.
Smith KC, and Hays JE.1968. The response of uracil-2-14C to Xirradiation under nitrogen and oxygen and to treatment with ascorbic acid. Radiat. Res 33:129–41. von Sonntag C. 1980. Free radical reactions of carbohydrates as studied by radiation techniques. Adv. Carbohydr. Chem. Biochem 37:7–77. von Sonntag C. Hagen U. Schön-Bopp A , and Schulte-Frohlinde D. 1981, Radiation-induced strand breaks in DNA: chemical and enzymatic analysis of end groups and mechanistic aspects. Adv. Radiat. Biol 9:109–42. von Sonntag C, and Thoms E. 1970, Strahlenchemie von Alkoholen XV γ-Radiolyse von Äthylenglykol in wässriger Lösung. Z. Naturf. b, 25:1405–7. Steenken S, Behrens G, and Schulte-Frohlinde D. 1974, Radiation chemistry of DNA model compounds. Part IV. Phosphate ester cleavage in radicals derived from glycerol phosphates. Int. J. Radiat. Biol 25:205–10. Stelter L, von Sonntag C, and Schulte-Frohlinde D. 1974, Radiation chemistry of DNA-model compounds. V. Phosphate elimination from ribose-5-phosphate after OH radical attack at C-4. Int. J. Radiat. Biol 25, 515-19;1975, Radiation chemistry of DNA-model compounds. VII. On the formation of 5-deoxy- d-erythro-pentos-4ulose and the identification of 12 further products from γ-irradiated aqueous solutions of ribose-5-phosphate. Z. Naturf. b, 30, 656–7; 1976, Phosphate ester cleavage in ribose-5-phosphate induced by OH radicals in deoxygenated aqueous solution. The effect ofFe(II) and Fe(III) ions. Int. J. Radiat. Biol 29:255–69. Taylor B, Greenstein JP, and Hollaender A . 1948. Effects of X-radiation on sodium thymus nucleate. Arch. Biochem 16:9–31. Ullrich M. and Hagen U. 1971, Base liberation and concomitant reactions in irradiated DNA solutions. Int J. Radiat. Biol. 19:507–17. Zegota H. Schuchmann MN. and von Sonntag C. 1984, Cyclopentylperoxyl and cyclohexylperoxyl radicals in aqueous solutions. A product and pulse radiolysis study. J. phys. Chem (in the press). Zegota H. and von Sonntag C. 1981, Radical-induced dephosphorylation of fructose phosphates in aqueous solution. Z. Naturf. b, 36:1331–7.
WEISS LECTURE †
Carbohydrate radicals: from ethylene glycol to DNA strand breakage* Clemens von Sonntag
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Max-Planck-Institut für Strahlenchemie, Stiftstr. 34–36, Mülheim/Ruhr, F.R. Germany It was in the late forties that Josef Weiss, Gabriel Stein and George Scholes started to investigate the radiation chemistry of nucleic acids (Scholes et al. 1949). It was pioneering work indeed because even the solvated electron was not yet fully established as an important species in the radiolysis of water. Nucleic acids were by no means commercially available and the knowledge of the chemistry of their constituents was limited, not to speak of their free-radical chemistry. A similar situation prevailed in the field of carbohydrate radiation chemistry where the first steps were soon afterwards to be made by both G. O. Phillips and N. K. Kochetkov (for reviews see Phillips 1961, 1971, Kochetkov et al. 1979, von Sonntag 1980). It was not before the late sixties that we became interested in the carbohydrate field as well, actually not so much in its own right but rather with an eye on the nucleic acids as our guiding star, where the sugar moiety must play an important role in the radiation-induced strand breaking process. This phenomenon was discovered by Taylor et al. (1948) and studied in some detail by Hagen (1964, 1967). Having had no experience in carbohydrate chemistry, it very soon became obvious to us that we would not be able to make any progress in this field if we started out with such relatively complex molecules, the analytics of which were not yet sufficiently well established. We therefore went back one further step and chose as the primary target of our investigation ethylene glycol, which represents one essential feature of carbohydrates, namely the vicinal glycol structure. From the product study (Seidler and von Sonntag 1969, von Sonntag and Thorns 1970) it soon became clear that the governing step of its free radical chemistry is a water elimination reaction (reaction (1)).
Abstract Radiation-induced DNA strand breakage results from the reactions of radicals formed at the sugar moiety of DNA. In order to elucidate the mechanism of this reaction investigations were first performed on low molecular weight model systems. Results from studies on deoxygenated aqueous solutions of ethylene glycol, 2-deoxy-D-ribose and other carbohydrates and, more relevantly, of D-ribose-5-phosphate have shown that substituents can be eliminated from the β-position of the radical site either proton and base-assisted (as in the case of the OH substituent), or spontaneously (as in the case of the phosphate substituent). In DNA the C(4’) radical undergoes strand breakage via this type of reaction. In the presence of oxygen the carbon-centred radicals are rapidly converted into the corresponding peroxyl radicals. Again, low molecular weights models have been investigated to elucidate the key reactions. A typical reaction of DNA peroxyl radicals is the fragmentation of the C(4’)-C(S’) bond, a reaction not observed in the absence of oxygen. Although OH radicals may be the important direct precursors of the sugar radicals of DNA, results obtained with poly(U) indicate that base radicals may well be of even greater importance. The base radicals, formed by addition of the water radicals (H and OH) to the bases would in their turn attack the sugar moiety to produce sugar radicals which then give rise to strand breakage and base release. For a better understanding of strand break formation it is therefore necessary to investigate in more detail the reactions of the base radicals. For a start, the radiolysis of uracil in oxygenated solutions has been reinvestigated, and it has been shown that the major peroxyl radical in this system undergoes base-catalysed elimination of O-2• , a reaction that involves the proton at N(l). In the nucleic acids the pyrimidines are bound at N(l) to the sugar moiety and this type of reaction can no longer occur. Therefore, with respect to the nucleic acids, pyrimidines are good models only in acid solutions where the O-2• elimination reaction is too slow to compete with the bimolecular reactions of the peroxyl radicals. Moreover, the long lifetime of the radical sites on the nucleic acid strand may allow reactions to occur which are kinetically of first order, and which cannot be studied in model systems at ordinary dose rates. It is therefore suggested to extend model system studies to low dose rates and to oligonucleo-tides. Such studies might eventually reveal the key reactions in radical-induced DNA degradation.
CHOH CH2OH → H2O CHO CH2
(1)
This interpretation was aided by e.s.r. studies where the process was. directly observed, at least at low pH (Bazhin et al. 1966, Buley et al. 1966, Livingston and Zeldes 1966). The water elimination reaction is both base- and acid-catalysed (Bansal et al. 1973) but at the low dose rate of 60Co γ-radiolysis it is fast enough to proceed to the extent that it governs the product distribution even in neutral solutions. The products shown in Table I are explained by the disproportionation and dimerization of the two radicals
†Given on the occasion of the presentation at the Association for Radiation Research meeting in Dublin, April 1984, of Weiss medals to Professors D. Schulte-Frohlinde and C. von Sonntag for their work on the radiation chemistry of DNA and its components. *This is a re-publication of an article published in a past International Journal of Radiation Biology issue (Von Sonntag, Clemens (1984) ‘Weiss Lecture’, International Journal of Radiation Biology, 46:5,507–519) for this special issue in honour of Professor Clemens von Sonntag.
(Received 12 June 1984)
416
Carbohydrate radicals 417
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Table I. γ-Radiolysis of ethylene glycol (101 m) in N2O-saturated aqueous solutions at various temperatures and a dose rate of ≃ 0.1 Gy s-1. Products and their G values according to von Sonntag and Thorns (1970). G value Products
0°C
20°C
80°C
Succindialdehyde Glycolaldehyde 2-Deoxytetrose Tetritol Acetaldehyde
1.9 0.9 0.3 0.15 0.5
1.7 1.1 0.25 0.15 1.2
2.3 0.8 0.13 0.03 6.4
shown in reaction (1). Also, favoured by elevated temperatures, low dose rate and high ethylene glycol concentration, acetaldehyde is formed via a chain reaction, the propagating step being reaction (2). CH2 CHO CH2OH CH2OH → CH3 CHO CHOH (2) CH2OH Analytical techniques were then refined (Dizdaroglu et al. 1972, 1974, 1977b) and it was eventually possible to fully analyse products from complex molecules such as 2-deoxyd-ribose (Hartmann et al. 1970) or d-glucose (cf. Table II), Table II. Products and their G values from the γ-radiolysis of deoxygenated, N2O-saturated or 4:1 N2O/O2-saturated aqueous solutions of d-glucose atadose rate of 0.18 Gy s1 at room temperature according to Dizdaroglu et al. (1975a) and Schuchmann and von Sonntag (1977). G value Product
N 2O
N2O/O2
d-Gluconic acid d-arabino-Hexos-2-ulose d-ribo-Hexos-3-ulose d-xylo-Hexos-4-ulose d-xylo-Hexos-S-ulose d-gluco-Hexodialdose 2-Deoxy-d-arabino-hexonic acid 5-Deoxy-d-threo-hexos-4-ulose 5-Deoxy-d-xylo-hexonic acid 2-Deoxy-d-erythro-hexos-5-ulose 5-Deoxy-d-xylo-hexodialdose 3-Deoxy-d-erythro-hexos-4-ulose 3-Deoxy-d-erythro-hexos-2-ulose 4-Deoxy-l-threo-hexos-5-ulose 6-Deoxy-d-xylo-hexos- 5 -ulose 2-Deoxy-d-erythro-hexos-3-ulose 4-Deoxy-d-threo-hexos-3-ulose d-Arabinose d-Arabinonic acid d-Ribose d-Xylose xylo-Pentodialdose 2-Deoxy-d-erythro-pentose d-Erythrose d-Erythronic acid l-Threose l-threo-Tetrodialdose 3-Deoxytetrulose 1,3-Dihydroxy-2-propanone d-Glyceraldehyde and glyceric acid Glyoxal Glyoxylic acid/glycolic acid Formaldehyde Formic acid d-Glucose consumption
0.15 0.15 0.10 0.075 0.18 0.22 0.95
0.90 0.90 0.57 0.50 0.60 1.55 absent absent absent absent absent absent absent absent absent absent absent 0.10
}
}
† Products identified (no
0.08
0.25 0.05 † † 0.01 absent 0.005 0.005 absent 0.04 0.01 absent 0.003 absent 0.02 0.03 absent ‡ ‡ ‡ ‡ 5.6
}
except for the dimer fraction, which in the case of deoxygenated solutions of glucose must contain more than a hundred different products. The discovery of the water elimination reaction, the knowledge of the nature of the products from this reaction, and the necessary analytics were timely, because soon it was recognized that this type of reaction is not restricted to the elimination of water, but, much more important for DNA research, that a similar reaction also prevails in phosphate esters, as shown by e.s.r. using glycerol phosphates as substrates (Samuni and Neta 1973, Steenken et al. 1974) and by product studies on d-ribose-5-phosphate (Stelter et al. 1974, 1975, 1976) as well as d-fructose phosphates (Zegota and von Sonntag 1981). Mechanistically, the phosphate elimination is somewhat different from the water elimination as it is already quite fast in neutral solutions, i.e. the reaction does not need to be speeded up by protons or hydroxide ions in order to become detectable by e.s.r. spectroscopy. In 1975 the first altered sugars resulting from radiationinduced DNA strand breakage were identified (Dizdaroglu et al. 1975 b). It had been postulated that radical cations might play a role as intermediates (cf. reaction (3)). Although at the time this was only a working hypothesis and other alternative mechanisms had to be considered (cf. von Sonntag et al. 1981) it was later shown that radical cations are the most likely intermediates (Behrens et al. 1978a, 1982); in special cases such species are sufficiently stable against water attack so that their e.s.r. spectra can be recorded (Behrens et al. 1978 b).
absent 0.08
}
absent 0.02 absent 0.20 absent absent 0.13 0.11 0.4 0.12 0.6 5.6
G values given). They are expected to be included in the G values of the other deoxyhexosuloses given in the table. ‡ Not determined, probably absent.
Armed with the knowledge of the principles of the carbohydrate free radical reactions—as the result of detailed studies of some further small model systems—we realized that
418
C. von Sonntag
sugars similar to those which were set free from the DNA on irradiation must also remain bound to DNA by the phosphate linkage. Hence techniques were developed to excise such lesions from irradiated DNA (Dizdaroglu et al. 1977a, Beesk et al. 1979). The altered sugars recognized so far, both free and bound to DNA, are shown in Table III. Not all of these alterations are linked with immediate strand breakage. Rather products 1, 2 and 12, and possibly 3, represent alkali-labile sites which turn into strand breaks upon alkali
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Table III. Alterations at the sugar moiety in the γ-radiolysis of DNA in deoxygenated and oxygenated solutions and site of precursor radicals. For references see text.
treatment. As shown in table III, some of the products appear only if the irradiation is carried out in the presence of oxygen. Besides these lesions which have been discovered at Mülheim, another lesion, brought about in the presence of oxygen, has been repeatedly reported, which is characterized by giving a positive 2-thiobarbituric acid (TBA) test (Krushinskaya and Shalnov 1967, Kapp and Smith 1970, Ullrich and Hagen 1971). The formation of glycolic acid bound to DNA 9 has also been reported (Henner et al. 1983). These observations would be compatible if in DNA a lesion of the type 13 was formed, similar to the products that have been suggested to be prominently related to bleomycin-induced strand breakage (Burger et al. 1980, Giloni et al. 1981). In the radiolytic system this TBA-active lesion still calls for a detailed analysis.
Because of the omnipresence of oxygen in nature and the sensitizing effect that oxygen exhibits in cellular systems, we became strongly interested in the effect of oxygen on these systems. Again, it had been J. Weiss who pioneered this field; he had also realised that one would have to investigate low molecular weight systems to obtain some insight into the general features. Being the modest and careful scientist he was, he considered it premature at the time of his investigations of the methane (Johnson and Weiss 1955) and ethylene systems (Clay et al. 1958) to put forward a mechanistic proposal to explain the products which he had observed. Since then, we have learned much about peroxyl radicals, and nowadays pulse radiolysis and more sophisticated analytical techniques allow us to reinvestigate these systems in more detail (Schuchmann and von Sonntag 1984, Piesiak et al. 1984) and to make mechanistic proposals. Reactions (13)—(22) represents our present knowledge of the decay of primary and secondary peroxyl radicals.
With a few exceptions (see below), peroxyl radicals decay bimolecularly. In the first step a short-lived tetroxide is formed (reaction (13)) which at low temperatures is in equilibrium with the peroxyl radicals (cf. reaction (14)) (Bennett et al. 1970) but which at elevated temperatures readily decomposes into products (Furimsky et al. 1980). Three concerted
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Carbohydrate radicals 419 processes have been recognized so far (reactions (15)–(17)), and one leading to molecular oxygen and two oxyl radicals (reaction (18)). The best known concerted process is the so-called Russell mechanism which leads to 02, alcohol and a carbonyl compound (reaction (15)) via a six-membered transition state (Russell 1957, Howard and Ingold 1968). The transition state of the second concerted process, the formation of H202 and two carbonyl compounds (reaction (16)) is likely to involve two five-membered rings (Bennett and Summers 1974, Bothe and Schulte-Frohlinde 1978, Schuchmann and von Sonntag 1979). It has been recently shown (Zegota et al. 1984, Piesiak et al. 1984) that the welldocumented (Howard 1973) fragmentation of the carboncarbon skeleton is also very likely to be due to a concerted process (reaction (17)). This reaction had earlier been postulated to involve free oxyl radicals (reaction (22)). Oxyl radicals carrying an a-hydrogen rearrange in water so quickly into the corresponding α-hydroxyalkyl radicals (reaction (19)) (Berdnikov et al. 1972, Gilbert et al. 1976, 1977, Schuchmann and von Sonntag 1981) that the fragmentation reaction (22) cannot compete effectively. The combination of two peroxyl radicals (reaction (20)) is usually of minor importance. Product-wise, the disproportionation reaction (21) cannot be distinguished from the Russell mechanism (reaction (15)). The α-hydroxyalkyl peroxyl radical formed by O2 addition to the α-hydroxyalkyl radicals can eliminate HO2 or, baseinduced, O-2• (reactions (23) and (24)).
Such radicals are not only formed in the course of the bimolecular decay of peroxyl radicals (Schuchmann and von Sonntag 1982) but more directly by OH-attack on alcohols and carbohydrates (Rabani et al. 1974, Ilan et al. 1976, Bothe et al. 1977, 1978b, 1983, for a review see Bothe et al. 1978a). For example, the major products observed in the radiolysis of d-glucose in oxygenated solutions (cf. Table II) are formed according to such a reaction (Schuchmann and von Sonntag 1977). In DNA there are no structural elements which, after OH attack and oxygen addition, would directly lead to the formation of α-hydroxyalkylperoxyl radicals. However, such radicals can also be formed by the sequence of the reactions (14)-(19). In competition there is almost always the fragmentation reaction (22). This type of reaction (or its concerted equivalent, reaction (17)) also appears to play a role in the formation of DNA lesions 2, 9, 10 and 11 (cf. Table III). With the exception of lesion 12 in all the altered sugars that have been observed in DNA so far (cf. Table III) an unaltered base is lost as well. This has led to an investigation of base release in a model system, poly(U) (Deeble and von Sonntag 1984) (Table IV). With this system it has been clearly shown that the release of unaltered base is induced by another base radical corroborating other evidence obtained in our institute (Lemaire et al. 1984) that base radicals induce strand breakage. The same reasoning put forward for deoxygenated systems can be extended to oxygenated ones
Table IV. Products and their G values in the g-radiolysis of N20 and N20/02 (4:1 )-saturated solutions of poly(U). G values N2O Strand breakage Base release (C) Oxygen uptake (d) Organic (hydro)peroxide (e) Hydrogen peroxide (e) Carbon dioxide (e) Osazone-forming compounds (e) Organic acid (pulse radiolysis) (f )
N2O/O2
2.3 (a) 2.9 — — n.d. absent 0.1 —
3.5 (b) 3.5 20 7 1.2 2.2 2.7 2.0
References: (a) Lemaire et al. (1984), (b) Lemaire cited by Schulte-Frohlinde and Bothe (1984), (c) Deeble and von Sonntag (1984), (d) Isildar et al. (1982), (e) Deeble and von Sonntag, unpublished results, (f ) Schulte-Frohlinde and Bothe (1984).
(Schulte-Frohlinde and Bothe 1984). If such a process were also to occur in DNA the sugar radicals ultimately responsible for strand breakage may well have base radicals as precursors. This brings us back to the free-radical chemistry of nucleobases, one of the early main research topics of Jo Weiss. Our group joined the research effort in this field some time ago (Campbell et al. 1974a, Hissung and von Sonntag 1978, 1979, Hissung et al. 1981, Schuchmann and von Sonntag 1983, Al-Sheikhly and von Sonntag 1983, Schuchmann et al. 1984b), and we have again experienced the old Newcastle stimulus in the cooperation with his old friend George Scholes (cf. Al-Sheikhly et al. 1984, Schuchmann et al. 1984a). In particular, the reinvestigation (for earlier work see Latarjet et al. 1962, Smith and Hays 1968, Peuzin et al. 1970, Ducolomb et al. 1971, 1973, Giroud et al. 1980) of the radiolysis of uracil in oxygenated solutions (Schuchmann et al. 1984b) has yielded interesting new information. The main (cf. Fujita and Steenken 1981) peroxyl radical in this system can undergo a base-induced O-2• -elimination (reaction (25)) which leads to isobarbituric acid and the glycols (reactions (26) and (27)) (Table V). In competition there is a bimolecular decay of which the typical products are isodialuric acid and formylhydroxyhydantoin (reactions (28)-(34)). Whereas at high pH, and also in neutral solutions at low dose rates, the O-2• -elimination reaction predominates, the bimolecular decay becomes prominent at low pH and in neutral solutions at high dose rates. Thus the simple model uracil is quite a complex system. The O-2• -elimination involves the proton at N(l). In the nucleic acids this proton is replaced by the sugar moiety, hence O-2• -elimination no longer occurs. Thus uracil irradiated in neutral solutions at low dose rates is not a perfect model Table V. γ-Radiolysis of N2O/O2-saturated uracil (2 104M) solutions under various pH conditions. G value Product 5,6-cis-Dihydroxy-5,6-dihydrouracil 5,6-trans-Dihydroxy-5,6-dihydrouracil Isobarbituric acid 1-N-Formyl-5-hydroxy-hydantoin Dialuric acid Isodialuric acid 5-Hydroxyhydantoin Unidentified product(s) Hydrogen peroxide Oxygen consumption Uracil consumption
pH3-0
pH6-5
pH100
0.6 0.5 0 1.6 0.9 0.1 0.4 0.9 n.d. n.d. 4.9
0.9 1.1 0.2 1.4 0.4 0.2 0.4 0.6 3.0 5.0 5.3
1.4 1.0 1.2 0.2 0.2 0.1 0.3 0.9 n.d. n.d. 5.2
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
420
C. von Sonntag
to study the fate of the pyrimidine peroxyl radical in nucleic acids. At first sight the uracil system studied in acid medium appears to be a better choice. Although this might well be so, one must always consider that in nucleic acids the radicals have very long lifetimes. Thus they can undergo reactions kinetically of (pseudo)first order which are not observed at the usual dose rates. We therefore suggest that model systems, e.g. small oligonucleotides, should be studied at very low dose rates. Such studies might reveal new reaction pathways hitherto not observed. In fact, base release from a nucleoside shows a pronounced dose rate effect at low pH (Fujita 1984). I like to think that Jo Weiss would agree wholeheartedly that we must continue to investigate model systems, that in fact it is impossible to sidestep the model systems, in order to arrive at a better understanding of the subject that we are primarily interested in: the radiation-induced changes of DNA within the cell.
References Al-Sheikhly M, Garner A , Hissung A , Schuchmann MN, Schuchmann, H.-P., Scholes, G., and von Sonntag, C. 1984. Radiolysis of dihydrouracil and dihydro-thymine in aqueous solutions contining oxygen; first- and second-order reactions of the rganic peroxyl radicals; the role of isopyrimidines as intermediates. J. chem. Soc. Perkin Trans. 2:601–8.
Al-Sheikhly M., and von Sonntag. C . 1983. γ-Radiolysis of 1,3-dimethyluracil in N2O-saturated aqueous solutions. Z. Naturf. b, 38:1622–9. Bansal KM, Grätzel M, Henglein A , and Janata E. 1973. Polarographic and optical absorption studies of radicals in the pulse radiolysis of aqueous solutions of ethylene glycol. J. phys. Chem., 77:16–19. Bazhin NM, Kuznetsov EV, Bubnov NN, and Voevodskii VV. 1966. Reaction of a hydrogen atom in the system H2O H2SO4 FeSO4. III. Reaction with saturated organic compounds, Kin. Katal. USSR (translated version) 7:643–5. Beesk F, Dizdaroglu M, Schulte-Frohlinde D, and von Sonntag C. 1979. Radiation-induced DNA strand breaks in deoxygenated aqueous solution. The formation of altered sugars as end groups. Int. J. Radiat. BioL 36:565–76. Behrens G, Bothe E, Eibenberger J, Koltzenburg G, and Schulte-Frohlinde D. 1978b. Nachweis eines Dialkoxyalken-Radikalkations in wässriger Lösung. Angew. Chem 90:639. Behrens G, Koltzenburg G, Ritter A , and Schulte-Frohlinde D. 1978a, The influence of protonation or alkylation of the phosphate group on the e.s.r. spectra and on the rate of phosphate elimination from 2-methoxyethyl phosphate 2-yl radicals. Int. J. Radiat. Biol 33:163–71. Behrens G, Koltzenburg G, and Schulte-Frohlinde D. 1982, Model reactions for the degradation of DNA-4’ radicals in aqueous solution. Fast hydrolysis of α-alkoxyalkyl radicals with a leaving group in β position followed by radical rearrangement and elimination reactions. Z. Naturf. c, 37:1205–27. Bennett JE, Brown DM, and Mile B. 1970. Studies by electron spin resonance of thereactions of alkylperoxy radicals. Part 2. Equilibrium between alkylperoxy radicals and tetroxide molecules. Trans. Faraday Soc 66:397–405.
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
Carbohydrate radicals 421 Bennett JE, and Summers R. 1974. Product studies of the mutual termination reactions of sec-alkylperoxy radicals: Evidence for non-cyclic termination. Can. J. Chem 52:377–9. Berdnikov VM, Bazhin NM, Fedorov VK, and Polyakov OV. 1972. Isomerization of the ethoxyl radical in aqueous solution. Kinet. Katal 13:86–7. Bothe E, Behrens G, and Schulte-Frohlinde D. 1977, Mechanism of the first order decay of 2-hydroxypropyl-2-peroxyl radicals and of O2• formation in aqueous solution. Z. Naturf. b, 32:86–9. Bothe E, Schuchmann MN, Schulte-Frohlinde D. and von Sonntag C. 1978a. HO2 elimination from O-hydroxyalkylperoxyl radicals in aqueous solution. Photochem. Photobiol 28:639–44. Bothe E, Schuchmann MN, Schulte-Frohlinde D. and von Sonntag C. 1983.Hydroxyl radical-induced oxidation of ethanol in oxygenated aqueous solutions. A pulse radiolysis and product study. Z. Naturf. b, 38:12–19. Bothe E, and Schulte-Frohlinde D. 1978. The bimolecular decay of the α-hydroxymethylperoxyl radical in aqueous solution. Z. Naturf. b, 33:786–8. Bothe E, Schulte-Frohlinde D, and von Sonntag C. 1978b. Radiation chemistry of carbohydrates. Part 16. Kinetics of HO2 elimination from peroxyl radicals derived from glucose and polyhydric alcohols. J. chem. Soc. Perkins Trans 2:416–20. Buley AL, Norman R.O.C, and Pritchett RJ. 1966. Electron spin resonance studies of oxidation. Part VIII. Elimination reactions of some hydroxyalkyl radicals. J. chem Soc. (B). 849-52. Burger RM, Berkowitz AR, Peisach J, and Horwitz SB. 1980. Origin of malondialdehyde from DNA degraded by Fe{II}. bleomycin. J. biol. Chem 255:11832–8. Campbell JM, Schulte-Frohlinde D, and von Sonntag C. 1974a. Quantum yields in the u.v. photolysis of 5-bromouracil in the presence of hydrogen donors. Photochem. Photobiol 20:465–7. Campbell JM, von Sonntag C, and Schulte-Frohlinde D. 1974b, Photolysis of 5-bromouracil and some related compounds in solution. Z. Naturf. b, 29:750–7. Clay PG, Johnson G.R.A , and Weiss J. 1958, Chemical action of ionising radiations in solution. Part XXI. The action of 60-Co-gammaradiation on aqueous solutions of ethylene at different pressures. J. chem. Soc 2175–82. Deeble DJ, and von Sonntag C. 1984. γ-Radiolysis of poly(U) in aqueous solution. The role of primary sugar and base radicals in the release of undamaged uracil from poly(U). Int. J. Radiat. Biol 46:247–260. Dizdaroglu M, Henneberg D, Schomburg G, and von Sonntag C. 1975a. Radiation chemistry of carbohydrates. VI. γ-Radiolysis of glucose in deoxygenated N2O-saturated aqueous solution. Z. Naturf. b, 30, 416–25. Dizdaroglu M, Henneberg D, and von Sonntag C. 1974. The mass spectra of TMS-ethers of deuterated polyalcohols. A contribution to the structural investigation of sugars. Org. Mass Spectrom 8:335–45. Dizdaroglu M, Henneberg D, von Sonntag C. and Schuchmann MN. 1977b. Mass spectra of trimethylsilyl di-O-methyloximes of adosuloses and dialdoses. Org. Mass Spectrom 12:772–5. Dizdaroglu M, Scherz H, and von Sonntag C. 1972. Strahlenchemie von Alkoholen-XVI. γ-Radiolyse von meso-Erythrit in wassriger Losung. Z. Naturf. b, 27:29–41. Dizdaroglu M, Schulte-Frohlinde D, and von Sonntag C. 1977a. γ-Radiolysis of DNA in oxygenated aqueous solution. Structure of an alkali-labile site. Z. Naturf, c, 32:1021–2. Dizdaroglu M, von Sonntag C, and Schulte-Frohlinde D. 1975b. Strand breaks and sugar release by γ-irradiation of DNA in aqueous solution. J. Am. Chem. Soc 97:2277–8. Ducolomb R, Cadet J, and Teoule R. 1971. Effect des rayon y sur l’uracil et l’acide uridylique en solution aqueuse aeree. C. R. Acad. Sci. Paris, Ser. D., 273, 2647–9; 1973, Effet du rayonnement gamma sur l’uracile en solution aqueuse aeree. Bull. Soc. Chim. France 1167–74. Fujita S, 1984. Radiolysis of nucleosides in aqueous solutions: base liberation by the baseattack mechanism. Int. J. Radiat. Biol 45: 371–7. Fujita S, and Steenken S. 1981. Pattern of OH radical addition to uracil and methyl-and carboxyl-substituted uracils. Electron transfer of OH adducts with N,N, N’ N’-tetramethyl-p-phenylenediamine and tetranitromethane. J. Am. Chem. Soc 103:2540–5. Furimsky E, Howard JA , and Selwyn J. 1980. Absolute rate constants for hydrocarbon autoxidation. 28. A low temperature kinetic electron spin resonance study of the self-reactions of isopropylperoxy and related alkylperoxyl radicals in solution. Can. J. Chem. 58:677–80. Gilbert BC, Holmes RGG, Lue, HAH. and Norman ROC. 1976. Electron spin resonance studies. Part L. Reactions of alkoxyl radicals
generated from alkyl-hydroperoxides and titanium (III) ion in aqueous solution. J. chem. Soc Perkins Trans. 2:1047–52. Gilbert BC, Holmes RGG, and Norman R.O.C. 1977. Electron spin resonance studies. Part LII. Reactions of secondary alkoxyl radicals. J. chem. Res. (S), 1. Giloni L, Takeshita M, Johnson F, Iden C, and Grollman AP. 1981. Bleomycin-induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J. biol. Chem. 256:8608–15. Giroud AM, Cadet J, and Ducolomb R. 1980. Etude par resonance magnetique nucleaire du carbone-13 des derives de degradation radio-induite de la thymine et del’uracile. N. J. Chim. 4:657–61. Hagen U. 1964. Untersuchungen über die Strahlenempfindlichkeit der Desoxyribo-nukleinsäure. II. Die Länge der Nukleotidketten nach Bestrahlung in vitro. Strahlen-therapie, 124:428–37;1967, Bestimmung von Einzel- und Doppelbrüchen in be-strahlter Desoxyribonucleinsäre durch die Molekulargewichtsverteilung. Biochim. biophys. Acta. 134:45–58. Hartmann V, von Sonntag C, and Schulte-Frohlinde D. 1970. γ-Radiolyse von 2-Desoxyribose in wässriger Lösung. Z. Naturf. 6, 25:1394–04. Henner WD, Rodriguez LO, Hecht SM, and Haseltine WA . 1983. Gamma-ray induced deoxyribonucleic acid strand breaks: 3’glycolate termini. J. biol. Chem. 258:711–13. Hissung A , and von Sonntag C. 1978, Radiolysis of cytosine, 5-methylcytosine and 2-deoxycytosine in deoxygenated aqueous solution. A pulse spectroscopic and pulse conductometric study of the OH adduct. Z. Naturf. b, 33, 321–8; 1979, The reaction of solvated electrons with cytosine, 5-methylcytosine and 2′-deoxycytidine in aqueous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study. Int. J. Radiat. Biol 35:449–58. Hissung A, von Sonntag C, Veltwisch D, and Asmus K.-D. 1981. The reaction of the 2’-deoxyadenosine electron adduct in aqueous solution. The effects of the radiosensitizer p-nitroacetophenone. A pulse spectroscopic and pulse conductrometric study. Int. J. Radiat. Biol. 39:63–71. Howard JA . 1973. Homogeneous liquid-phase autoxidations. Free Radicals, edited by J. K. Kochi, Vol. 2 (New York: Wiley), pp. 3–62. Howard JA , and Ingold KU. 1968. The self-reaction of sec-butylperoxy radicals. Confirmation of the Russell mechanism. J. Am. chem. Soc. 90:1056–8. Ilan Y, Rabani J, and Henglein A . 1976. Pulse radiolytic investigations of peroxy radicals produced from 2-proponal and methanol. J. phys. Chem. 80:1558–65. Isildar M, Schuchmann MN, Schulte-Frohlinde D, and von Sonntag C. 1982. Oxygen uptake in the radiolysis of aqueous solutions of nucleic acids and their constituents. Int. J. Radiat. Biol, 41: 525–33. Johnson GRA , and Weiss J. 1955. The formation of methyl hydroperoxyde from methane irradiated by X-rays (200 kV) in aqueous solution in the presence of dissolved oxygen. Chem. Ind, 358-9. Kapp DS, and Smith KC. 1970. Chemical nature of chain breaks in DNA by X-irradiation in vitro. Radiat. Res. 42:34–49. Kochetkov NK, Kudrjashov LI, and Chlenov MA . 1979. Radiation Chemistry of Carbohydrates, translated by G. O. Phillips (Oxford: Pergamon Press). Krushinskaya NP, and Shalnov MI. 1967. The nature of breaks of the DNA chain by irradiation of its water solutions. Radiobiology 7:36–45. Latarjet R, Ekert B, Apelgot S, and Rebeyrotte N. 1962. Etudes radiobiochimiques sur l’ADN. J. chim. Phys., 58:1046–57. Lemaire DGE, Bothe E, and Schulte-Frohlinde D. 1984. Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals. Int. J. Radiat. Biol 45:351–8. Livingstone R, and Zeldes H. 1966. Paramagnetic resonence study of liquids during photolysis. III. Aqueous solutions of alcohols with hydrogen peroxide. J. Am. chem. Soc 88:4333–6. Peuzin MC, Cadet J, Polverelli M, and Teoule R. 1970. Parabanic acid and 5-hydroxy hydantoin upon y-irradiation of aerated aqueous solutions of uracil. Biochim. biophys. Acta 209:573–4. Phillips GO, 1961. Radiation chemistry of carbohydrates. Adv. Carbohydr. Chem. Biochem., 16, 13–58; 1971, Effects of ionizing radiations on carbohydrate systems. Radiat. Res. Rev 3:335–51. Piesiak A , Schuchmann MN, Zegota H. and von Sonntag C. 1984. (β-Hydroxyethylperoxyl radicals: A study of the y-radiolysis and pulse radiolysis of ethylene in oxygenated aqueous solutions. Z. Naturf. b, 39, (in the press). Radani J, Klug-Roth D, and Hengi.ein A . 1974, Pulse radiolytic investigations of OHCH2O2 radicals. J. phys. Chem 78:2089–93.
Int J Radiat Biol Downloaded from informahealthcare.com by Northern Illinoise university Libraries on 01/28/15 For personal use only.
422
C. von Sonntag
Russell GA , 1957, Deuterium-isotope effects in the autoxidation of aralkyl hydrocarbons. Mechanism of the interaction of peroxy radicals. J. Am. chem. Soc 79:3871–7. Samuni A , and Neta P. 1973, Hydroxyl radical reactions with phosphate esters and the mechanism of phosphate cleavage. J. phys. Chem 77:2425–9. Scholes G, Stein G, and Weiss J. 1949, Action of X-rays on nucleic acids. Nature, Lond 164:709–10. Schuchmann H.-P, and von Sonntag C. 1981. Photolysis at 185 nm of dimethyl ether in aqueous solution: involvement of the hydroxymethyl radical. J. Photochem 16:289–95;1984, Methylperoxyl radicals: a study of the γ-radiolysis of methane in oxygenated solutions. Z. Naturf. b, 39:217–21. Schuchmann MN, Al-Sheikhly M, von Sonntag C, Garner A. and Scholes G. 1984a. The kinetics of the rearrangement of some isopyrimidines to pyrimidines studied by pulse radiolysis. J. chem. Soc. Perkin Trans. 2 (in the press). Schuchmann MN, and von Sonntag C. 1977. Radiation chemistry of carbohydrates. Part 14. Hydroxyl radical-induced oxidation of d-glucose in oxygenated aqueous solution. J. chem. Soc. Perkin Trans 2:1958-63; 1979, Hydroxyl radical-induced oxidation of 2-methyl-2propanol in oxygenated aqueous solution. A product and pulse radiolysis study. J. phys. Chem 83:780-4;1982, Hydroxyl radical induced oxidation of diethylether in oxygenated aqueous solution. A product and pulse radiolysis study. J. phys. Chem 86,1995–2000;1983, The radiolysis of uracil in aqueous solutions. A study by product analysis and pulse radiolysis. J. chem. Soc. Perkin Trans 2:1525–31. Schuchmann MN, Steenken S, Wroblewski J, and von Sonntag C. 1984b. Site of OH attack on dihydrouracil and some of its methyl derivatives. Int. J. Radiat. Biol 46:225–232. Schulte-Frohlinde D, and Bothe E. 1984. Identification of a major pathway of strandformation in poly U induced by OH radicals in presence of oxygen. Z. Naturf. c, 39:315–19. Seidler F, and von Sonntag C. 1969. Strahlenchemie von Alkoholen– VIII. Die Acetaldehydbildung bei der γ-Radiolyse wässriger, N2Ogesättigter L ösungen von Äthylenglykol. Z. Naturf. b, 24:780–1.
Smith KC, and Hays JE.1968. The response of uracil-2-14C to Xirradiation under nitrogen and oxygen and to treatment with ascorbic acid. Radiat. Res 33:129–41. von Sonntag C. 1980. Free radical reactions of carbohydrates as studied by radiation techniques. Adv. Carbohydr. Chem. Biochem 37:7–77. von Sonntag C. Hagen U. Schön-Bopp A , and Schulte-Frohlinde D. 1981, Radiation-induced strand breaks in DNA: chemical and enzymatic analysis of end groups and mechanistic aspects. Adv. Radiat. Biol 9:109–42. von Sonntag C, and Thoms E. 1970, Strahlenchemie von Alkoholen XV γ-Radiolyse von Äthylenglykol in wässriger Lösung. Z. Naturf. b, 25:1405–7. Steenken S, Behrens G, and Schulte-Frohlinde D. 1974, Radiation chemistry of DNA model compounds. Part IV. Phosphate ester cleavage in radicals derived from glycerol phosphates. Int. J. Radiat. Biol 25:205–10. Stelter L, von Sonntag C, and Schulte-Frohlinde D. 1974, Radiation chemistry of DNA-model compounds. V. Phosphate elimination from ribose-5-phosphate after OH radical attack at C-4. Int. J. Radiat. Biol 25, 515-19;1975, Radiation chemistry of DNA-model compounds. VII. On the formation of 5-deoxy- d-erythro-pentos-4ulose and the identification of 12 further products from γ-irradiated aqueous solutions of ribose-5-phosphate. Z. Naturf. b, 30, 656–7; 1976, Phosphate ester cleavage in ribose-5-phosphate induced by OH radicals in deoxygenated aqueous solution. The effect ofFe(II) and Fe(III) ions. Int. J. Radiat. Biol 29:255–69. Taylor B, Greenstein JP, and Hollaender A . 1948. Effects of X-radiation on sodium thymus nucleate. Arch. Biochem 16:9–31. Ullrich M. and Hagen U. 1971, Base liberation and concomitant reactions in irradiated DNA solutions. Int J. Radiat. Biol. 19:507–17. Zegota H. Schuchmann MN. and von Sonntag C. 1984, Cyclopentylperoxyl and cyclohexylperoxyl radicals in aqueous solutions. A product and pulse radiolysis study. J. phys. Chem (in the press). Zegota H. and von Sonntag C. 1981, Radical-induced dephosphorylation of fructose phosphates in aqueous solution. Z. Naturf. b, 36:1331–7.
