INT .
J.
RADIAT . BIOL .,
1992,
VOL .
62,
NO .
5, 517-526
Ethanol radical-induced protein-DNA crosslinking . A radiolysis study H . SCHUESSLER*t, G . SCHMERLER-DREMELt, J . DANZERf and E . JUNG-KORNERt
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(Received 20 March 1992; revised 9 June 1992; accepted 12 June 1992)
Abstract . Aqueous solutions of double-stranded DNA from calf thymus and bovine serum albumin (BSA) were irradiated at pH 7 under N 2 0 and N 2 in the presence of 10 -1 mol dm -3 ethanol, which was partly 14 C-labelled . Ethanol protects DNA from strand breakage by scavenging OH radicals, but ethanol radicals induce protein-DNA crosslinks . Ethanol radicals react readily with BSA mainly by addition . They react also with double-stranded DNA, but produce crosslinking only very slowly . Based on these results the following mechanism is proposed : ethanol radicals bind to BSA producing protein radicals which become crosslinked to DNA .
alteration is responsible for the loss of biological activity, further work has now been done to understand in which way secondary radicals react with DNA and proteins . Though with t-butanol radicals a higher yield of protein-DNA crosslinks is obtained than with ethanol radicals, this study has been carried out with ethanol, because 14C-labelled tbutanol was not available .
2. Materials and methods
1. Introduction Ethanol is usually considered to be a good protector against radiation-induced damage, as shown by various experiments in cells (e.g. Roots and Okada 1972, Ewing 1976) and with biopolymers (e.g. Schuessler and Hartmann 1985) . Protection is caused by ethanol scavenging OH and H radicals under the assumption that the alcohol radicals formed are inert . There are now several publications indicating a considerable reactivity of such secondary radicals with biomolecules or biological systems as ribosomes or chromatin (e.g. Nabben et al. 1983, Singh and Bishop 1983, Singh and Vadasz 1984, Herskind and Westergard 1988) . These reactions cause loss of biological activity, but the molecular mechanisms are uncertain . It was demonstrated in vitro that OH scavenging prevents strand breakage of double-stranded DNA (Washino and Schnabel 1982, Schuessler and Hartmann 1985) . However, we showed that secondary radicals formed by OH scavenging can also cause crosslinking of proteins to DNA (Schuessler and Jung 1989), although it was assumed that in cells protein-DNA crosslinks were induced by OH radicals (Oleinick et al . 1986) . Since it is possible that this *Author for correspondence . tInstitut fur Radiologie der Universitat Erlangen-Niirnberg, 8520 Erlangen, Germany . This work was supported by the Deutsche Forschungsgemeinschaft .
2.1 . Materials Highly polymerized double-stranded DNA from calf thymus (average m .w. - 2 . 107 D) was from Sigma Chemie GmbH (Munchen), 14C-ethanol from DuPont (Dreieich), lyophilized bovine serum albumin (BSA) from Boehringer GmbH (Mannheim), Sepharose from Pharmacia (Freiburg), and Aquasafe from Zinsser Analytic (Frankfurt/M .) . Other chemicals were of analytical reagent grade from Merck (Darmstadt) . Solutions were made with reagent-grade water purified by deionization, reverse osmosis, and a Milli-Q-system .
2.2 . Irradiation Radiolysis was carried out in 1 x 10 -2 moldm -3 phosphate buffer -3 )at pH 7 containing ethanol (1 x 10 -1 mol dm partly labelled with 14C . DNA (1 mg) was dis(37 MBq dm -3 =1 uCi/ml) solved in 10 ml of this solution by incubation in a refrigerator for 8 h and slow stirring for 30 min . BSA concentration was 1 .0 mg/ml . Irradiation was performed as described previously (Schuessler et al. 1987) with a Stabilipan X-ray machine (Siemens, Erlangen) with 200 kV, 20 mA, and a 2 mm aluminium filter . The dose rate was 0 .5 Gy s - 1 as estimated by Fricke dosimetry .
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
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2 .3 . Gel filtration Biomolecules and ethanol were separated at 4°C by gel filtration on Sepharose CL-2B (100 x 2 .5 cm) with 1 x 10 -2 mol dm -3 phosphate buffer containing 0 .02% NaN 3 at pH 7 . The eluates were collected in 3 .0 ml fractions in which the nucleic acid content was determined by the absorbance at 260 nm ; I ml per fraction was combined with 10 ml Aquasafe, and dpm were determined with a liquid scintillation counter (Primo LKB, Finland) . Protein was assayed as reported previously (Schuessler et al. 1987) with Coomassie Brilliant Blue G-250 according to the method of Sedmak and Grossberg (1977) . If BSA had to be separated from ethanol, gel filtration was carried out on Sepharose 6B (90 x 2.5 cm), and the concentration of BSA was determined by measuring the absorbance at 280 rim . Further information on the methods may be found in a thesis (Danzer, 1992) . 3. Results Previous work (Schuessler and Jung 1989) showed that reactions of secondary radicals, e .g. alcohol radicals, can cause protein-DNA crosslinks . To elucidate the mechanisms of these reactions, partly 14C-labelled ethanol was used ; DNA and BSA in a ratio of 1 :10 by mass were irradiated in the presence of I x 10 -1 mol dm -3 ethanol in 1 x 10 -2 mol dm -3 phosphate buffer pH 7 .0 under N 2 0 or N 2 . The biopolymers and ethanol were separated by conventional gel filtration on Sepharose CL-2B (Figure 1) . Highly polymerized DNA was eluted in the void volume of this column, while BSA was retarded and was found in fractions 70-100 simultaneously with ethanol . With increasing dose the amount of BSA and ethanol eluting with DNA in the void volume increased . The total amount of protein in the chromatogram was taken as 100% and the percentage of protein bound to DNA was determined . The yield of crosslinked ethanol could be measured because the ratio of 14 C- to 12 C-ethanol was known . Crosslinking of BSA and of ethanol is shown as a function of dose for radiolysis under N 2 (Figure 2) and under N 2 0 (Figure 3) . In both cases the yield of crosslinked BSA increased linearly with dose, resulting for this dose range in a constant yield of 1 . 6 ±0.3nmolJ -1 under N 2 and 0 . 7±0. l nmolJ -1 under N 2 0. Since under N 2 0 the hydrated electrons are converted to OH radicals and then to ethanol radicals, it is obvious that electrons are more effective in crosslinking BSA to DNA than ethanol radicals . This result confirms earlier findings
et al .
obtained with a nitrocellulose filter binding assay and HPLC (Schuessler and Jung 1989) . The yields of crosslinked ethanol are the same in the range of error under N 2 and N 2 0. The measured values for bound ethanol as a function of the dose follow approximately a polynomial of second order . The yields for ethanol crosslinked to DNA increase linearly with the dose (Figure 8) . To answer the question whether ethanol radicals react with double-stranded DNA, radiolysis of DNA in the presence of 1 x 10 -1 mol dm -3 ethanol was investigated . An obvious effect of ethanol is protection against double-strand breakage by scavenging all OH radicals (Figure 4) . Very high doses are necessary to show the degradation of DNA under these conditions . Since no difference was observed by changing from N 2 to N 2 0, the conclusion may be that ethanol radicals have the same low efficiency causing strand breaks as hydrated electrons . For comparison, DNA degradation by radiolysis without ethanol (Schuessler and Hartmann 1985) is shown (Figure 4) . In addition to this weak degradation ethanol radicals become crosslinked to doublestranded DNA (Figure 5) . The yield of ethanol eluted with DNA increased linearly with the dose, G=5. 2±0.8nmolJ -1 under N 2 or N 2 0. To determine how ethanol radicals react with BSA under these conditions, BSA (1 mg/ml) was irradiated in the presence of 1 x 10 -1 mol dm -3 ethanol partly labelled with 14C and analysed by gel filtration on Sepharose 6B . On this gel BSA could be separated from unbound ethanol . The monomer BSA was found in fractions 74-83, while the elution of unbound ethanol started at fraction 94 (Figure 6) . With increasing dose the amount of ethanol bound to BSA increased ; simultaneously, BSA was eluted earlier than monomet BSA . The loss of monomer BSA is not caused by unfolding of protein but by aggregation as shown by SDS-polyacrylamide gel electrophoresis in previous work (Schuessler and Freundl 1983) . The yield for the loss of monomer BSA was 2. 3 nmo1J -1 at pH 6 . 3 and 0 . 5 nmolJ -1 at pH 8 .2. The elution patterns (Figure 7) show that after 450 Gy the ratio of ethanol to BSA is about 3 :1 and is constant in the peak which indicates a rather homogeneous radiation product . With increasing dose, aggregates are formed to which more ethanol molecules are crosslinked than to the monomer BSA . The yield for crosslinking of ethanol to BSA decreased with the dose (Figure 8) . Under N 2 0 with 900 Gy, G = 97 nmol J -1 was found, but at 2400 Gy it was only 78 nmol J -1 . Under N 2 the yields were lower than under N 2 0; for _1. the same doses they were 76 and 41 nmol J A comparison of all measured yields (Figures 5 and 8) showed that the lowest was found for cross-
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Ethanol radicals with BSA and DNA
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Elution pattern of DNA and BSA irradiated together in the presence of 1 x 10 -1 mol dm -3 ethanol under N2 (Sepharose CL-2B) .
linking of BSA to DNA . The yields for crosslinking of ethanol to DNA and BSA to DNA were constant in the dose range up to 5000 Gy . Ethanol radicals are much more efficient in crosslinking to BSA than to DNA . At low doses under N 2 0 the yields are about 19 times higher for protein than for DNA, but they
decrease with the dose . On the other hand, crosslinking of ethanol to DNA in the presence of BSA increases with the dose and was found at 5000 Gy to be 20 nmol J -1 , which would mean that to each BSA which was crosslinked to DNA on average 30 ethanol molecules were bound .
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BSA and ethanol bound to undegraded DNA after radiolysis of BSA and DNA in the presence of 1 x 10 -1 mol dm -3 ethanol under N 2 .
rather slow . Since secondary radicals induce protein-DNA crosslinks, the assumption is that ethanol radicals add to BSA and that these protein radicals become crosslinked to DNA . Ethanol radicals are formed by reactions of OH radicals (koH •+ E1h=1 .8 x 10 9 mol -1 dm3 s-1 ) and by H radicals (kH .+Eth=1 .6 x 10 7 mol -1 dm3 s -1 ) (Dorfman and Adams 1973) . These two primary radicals produce mainly a-hydroxyalkyl radicals : 84% are a-, 13% fl- and 2 . 5% are 0 radicals (Asmus et al . 1973) . These radicals decay by association and disproportionation reactions with a second-order
4. Discussion This work confirms that ethanol protects doublestranded DNA from strand breakage . It provides further evidence that ethanol radicals are not inert but react with biopolymers and induce different modifications . Ethanol radicals react to a great extent with proteins, as shown in this and previous work (Schuessler 1975, 1981, Schuessler et al . 1976, Schuessler and Freundl 1983, Puchala and Schuessler 1986), mainly by addition . The reaction of ethanol radicals with double-stranded DNA is
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BSA and ethanol bound to undegraded DNA after radiolysis of BSA and DNA in the presence of 1 x 10 -1 mol dm -3 ethanol under N 2 0 .
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30 20 without ethanol
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dose (Gy) Figure 4 . Radiolysis of DNA without BSA : double-stranded DNA eluted in the void volume as a function of the dose .
rate constant (2k=2 . 3 x 10 9 M -t s -1 ; Simic et al. 1969) . Ethanol can react with organic molecules (RH) in three ways :
(iii)
by addition R+CH 3 (~HOH-*R(CH 3 )CHOH
4.1 . Radiation-induced reactions of ethanol and BSA
(i) by electron transfer RH + CH 3 CHOH,RH' + CH 3 CHO + H + (ii) by H-abstraction RH + CH3 CHOH--•R ' + CH 3 CH 2 OH
Reaction of ethanol radicals with BSA leads to crosslinking of ethanol to BSA and to aggregation of
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Figure 5 . Crosslinking ethanol to DNA by radiolysis of DNA in the presence of 1 x 10 -1 mol dm -3 ethanol without BSA under N 2 or N 2 0 . Upper : G-values (bound molecules/ 100 eV) . (G=0. 1 molec/ 100 eV corresponds to a yield of 10 . 4nmolJ -1 .) Lower : µmoles of bound ethanol .
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Elution patterns of BSA from Sepharose 6B, before and after radiolysis in the presence of 1 x 10
the protein . At 900 Gy the yield for crosslinking under N 20 was 90nmolJ -1 (Figure 8) and under N2 70nmo1J -1 , indicating that 15 and 26%, respectively, of all ethanol radicals become bound to BSA . With increasing dose the yields decrease . A plausible explanation is that amino acid residues which react fast with ethanol radicals decrease with dose .
-1 mol
dm -3 ethanol .
Under comparable conditions ethanol crosslinking to two other proteins have been measured . With ribonuclease the yield for crosslinking under N 20 with 5kGy is 60 and under N 2 50nmolJ -1 (Schuessler 1981) . With haemoglobin the yields are similar, 54 under N 20 and 48nmolJ -1 under N 2 (Puchala and Schuessler 1986), but constant up to a dose of 4 .8 kGy .
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Ethanol radicals with BSA and DNA
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Elution profile of BSA and the ratio of bound ethanol molecules per BSA molecule after radiolysis of BSA in the presence of 1 x 10 -1 moldm -3 ethanol under N2 0 -
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dose (Gy) G-values (bound molecules/ IOOeV) for crosslinking of ethanol, BSA, and DNA after irradiation under N 2 0 at pH 7 . = crosslinking between ethanol and BSA in the presence of DNA ; O = crosslinking between ethanol and DNA in the presence of BSA ; O =crosslinking between BSA and DNA in the presence of ethanol. (G=1 molec/100 eV corresponds to a yield of 104 nmol J -1 . )
Figure 8 .
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Pulse radiolysis studies (Schuessler and Davies 1983) of secondary radicals and BSA gave evidence that formate radicals with a redox potential E°(C0 2/CO2 ) = -2 . 0 V can transfer electrons to the disulfide groups in BSA . In contrast, ethanol radicals with a redox potential E°(Eth/Eth')= -1 .2 V (Breitenkamp et al . 1976) have a low rate and yield for electron transfer, so that with 1 .5 x 10 - 5 mol dm - 3 BSA, the concentration used in this work, only 1-2% can react in this way . This result was also confirmed by studying radiation products after steady state radiolysis of BSA and secondary radicals (Schuessler and Freundl 1983) . While the reaction of formate radicals yields aggregates of BSA by the formation of intermolecular S-Sbridges, the reaction of ethanol radicals leads only to non-reducible aggregates, indicating that ethanol radicals did not transfer electrons to cystine in BSA . It was previously assumed that aggregation can be induced by H-abstraction by ethanol radicals : CH3HOH + ProtH-+CH 3CH2OH + Prof Prof + Prot'--- Prot - Prot Prot' + Prot-+Prot - Prof However, our experimental results show that crosslinking of ethanol to BSA is more efficient than aggregation. Also it is obvious that more ethanol molecules per BSA molecule are bound to BSA aggregates than to monomer BSA (Figure 7) . For these reasons it is now assumed that the main reaction under N 20 is the addition of ethanol radicals to BSA molecules . CH3HOH+Prot-*(Prot-CHOH)' CH3 The protein radicals can react further with ethanol radicals or can produce aggregation . In accordance with all studied proteins a higher percentage of ethanol become bound to BSA under N 2 than under N20. A plausible explanation is that hydrated electrons form BSA radicals which then react with ethanol radicals : eay + Prot-+Pros' CH 3 CHOH + Prof -- Prot - CHOH CH3 4.2. Radiation-induced reactions of ethanol and DNA In this work crosslinking of ethanol to doublestranded DNA could be detected after radiolysis
under anaerobic conditions . The yield under N 2 0 as well as under N 2 is 5nmolJ -1 (Figure 5), which means that only 0 .8 and 1 .5%, respectively, of all ethanol radicals become crosslinked . It is known that alcohol radicals react with nucleobases. Addition products between ethanol and thymine as well as with dimethylthymine and thymidine have been found (Brown et al . 1966, Zarebska and Shugar 1972, Cadet et al . 1981) . In more recent work (Schuchmann et al . 1986) reactions of methanol radicals with dimethyluracil and dimethylthymine have been studied . The analysis of the radiation products show that the hydroxyalkyl radicals add with a very high specificity to the C(6) position with a yield of 80 nmol J -1 at a dose rate of 0. 19 Gy s -1 . This yield can be increased by reducing the dose rate, because the rate constant for hydroxymethyl radicals with pyrimidine is only about 1 x 104 mo1 -1 dm3 s -1 . While about two-thirds of the hydroxymethyl radicals add to dimethyl thymine, one-third abstracts an H-atom from the methyl group . Both thymidine radicals can react further with a second ethanol radical or undergo dimerization . The a-hydroxyalkyl radicals also react with purines forming compounds alkylated at C(8) (Steinmaus et al. 1969, 1971) . Single-stranded DNA from 0 X 174 can be inactivated by iso-propanol radicals (Nabben et al . 1983) . For this reaction a rate constant of 7.4 x 10 3 mol -1 dm3 s-1 was measured . Experiments with light-induced reaction of a-hydroxyalkyl radicals with single-stranded DNA showed that the rate is an order of magnitude slower than with nucleobases and an additional order of magnitude lower with double-stranded DNA (Livneh et al. 1982) . To our knowledge there is no publication on irradiation-induced reactions of ethanol with double-stranded DNA . The low yield for crosslinking of ethanol to DNA, which was found in this work, is consistent with data from the relevant literature . Accordingly, it is assumed that crosslinking is caused by addition and H-abstraction . Since the yield under N 2 0 and N 2 is the same, DNA radicals produced by hydrated electrons seem to react also with ethanol radicals . Furthermore, it was observed that ethanol radicals and ethanol radicals with hydrated electrons cause a slight degradation of DNA observable only with very high doses (Figure 4) and almost negligible in comparison with degradation caused by OH radicals . Strand breaking of DNA by ethanol radicals may be induced by Habstraction from DNA .
525
Ethanol radicals with BSA and DNA 4 .3 . Radiation-induced reactions of ethanol with BSA and
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DNA
The main reason for this work was to investigate the induction of protein-DNA crosslinks by secondary radicals . Under conditions of complete scavenging of OH radicals by ethanol, the yield for crosslinking BSA to DNA is still 1-5 under N 2 and 0. 7nmolJ -I under N 2 0 (Figure 8) . These values are similar to those published previously which were 1 . 5 and 0.4nmo1J -I , respectively (Schuessler and Jung 1989) . Simultaneously, with BSA, ethanol molecules become bound to DNA, in fact rather efficiently, increasing with dose from 9 to 30 ethanol per BSA . Furthermore, no aggregation of BSA could be found under this condition . The prevention of aggregation of BSA by the presence of DNA has previously been observed, when crosslinks were induced by primary radicals. It was assumed that there is an electrostatic interaction between DNA and BSA which contributes to the yield of crosslinks . This assumption could be confirmed by lowering the pH which leads to stronger interaction and a higher yield of crosslinks between BSA and DNA (Schuessler et al . 1987) . If the ratio of BSA : DNA is 10 :1 by mass and the dose 440 Gy under N 2 , there are 1500 BSA molecules crosslinked to one DNA molecule with a molecular weight of 2. 107 D . Considering the dimensions of the two biopolymers, between 700 and 2500 BSA molecules can become attached on the two sides of one DNA molecule . When the ratio BSA :DNA is increased over 10 :1 by mass, then the yield of BSA-DNA crosslinks decreases and aggregations of BSA occurs again (Goetzinger 1992) . This work shows that the efficiency of ethanol radicals is much higher in adding to BSA than to DNA. Therefore, if BSA and DNA with a ratio 10 :1 are in solution, ethanol radicals will attack the protein exclusively, producing protein radicals, mainly by addition and H-abstraction . These protein radicals react further with other ethanol radicals or with DNA, but not with another BSA molecule because they are not free to diffuse . If BSA alone was irradiated with ethanol under N 2 0 with a dose of 2400 Gy, the ratio of ethanol crosslinked to BSA is 12.5 :1 on average, while with the same dose this ratio is 24 :1, if BSA is bound to DNA . This high binding ratio of ethanol to BSA reflects the lower chance of the BSA radical participating in a crosslinking reaction to DNA than to an additional ethanol radical . This is similar to the aggregation of proteins induced by ethanol radicals . Aggregates also have a higher amount of crosslinked ethanol than the separated monomer (Figure 7) .
Under N 2 the yield for crosslinking BSA to DNA is twice as great as under N 2 0 . This is because the hydrated electrons are more efficient in producing protein radicals to crosslink with DNA, but the ratio Eth :BSA is lower than under N 2 0. At 2400 Gy this ratio is 8, while if radiolysis is carried out in the absence of DNA it is about 7 . This study indicates that ethanol radicals add to proteins in preference to DNA, forming protein radicals which become crosslinked to DNA . Accordingly, it has to be assumed that in cells the primary target by secondary radicals is not DNA but more probably proteins, and that besides strand breaks protein-DNA crosslinks may cause loss of biological activity . Furthermore, this work illustrates how complex radiation-induced reactions between biopolymers in a solution become with the addition of only one substance and that further investigations are required to understand the molecular radiation biology of the cell .
Acknowledgements The authors thank the Deutsche Forschungsgemeinschaft for financial support of this work .
References AsMus, K .-D ., MOCKEL, H . and HENGLEIN, A ., 1973, Pulse radiolytic study of the site of OH' radical attack on aliphatic alcohols in aqueous solution . Journal of Physical Chemistry, 77, 1218-1221 . BREITENKAMP, M ., HENGLEIN, A . and LILIE, 1976, Mechanism of the reduction of lead ions in aqueous solution . Berichte der Bundesgesellschaft fur Physikalische Chemie, 80, 973-979 . F ., 1966, Thymine BROWN, P. E ., CALVIN, M . and NEWMARK, addition to ethanol : induction by gamma irradiation .
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by OH scavengers against radiation-induced inactivation of isolated transcriptionally active chromatin : the influence of secondary radicals . Radiation Research, 114, 28-41 . LIVNEH, E ., TEL-OR, S ., SPERLING, J . and ELAD, D., 1982, Light-induced free-radical reactions of purines and pyrimidines in deoxyribonucleic acid . Effect of structure and base sequence on reactivity . Biochemistry, 21, 3698-3703 . NABBEN, F . J ., STROM, H . A . VAN DER, and LOMAN, H ., 1983, Inactivation of biologically active DNA by isopropanol and formate radicals . International Journal of Radiation Biology, 43, 495-504 . OLEINICK, N . L ., CHID, S ., RAMAKRISHAN, N . and XUE, L ., 1986, DNA-protein crosslinks : new insights into their formation and repair in irradiated mammalian cells . In : Mechanisms of DNA Damage and Repair . Edited by : M . G . Simic, L . Grossmann and A . C . Upton, pp . 181-192 (Plenum Press, New York and London) . PUCHALA, M . and SCHUESSLER, H ., 1986, Radiation-induced binding of methanol, ethanol and l-butanol to haemoglobin . International Journal of Radiation Biology, 50, 535-546 . ROOTS, R . and OKADA, S ., 1972, Protection of DNA molecules of cultured mammalian cells from radiation-induced single-strand scissions by various alcohols and SH compounds . International Journal of Radiation Biology, 21, 329-342 . SCHUCHMANN, H .-P ., WAGNER, R . and SONNTAG, C . VON, 1986, The reactions of the hydroxymethyl radical with 1,3dimethyluracil and 1,3-dime thylthymine . International Journal of Radiation Biology, 50, 1051-1068 . SCHUESSLER, H ., 1975, Effect of ethanol on the radiolysis of ribonuclease . International Journal of Radiation Biology, 27, 171-180 . SCHUESSLER, H ., 1981, Reactions of ethanol and formate radicals with ribonuclease A and bovine serum albumin in radiolysis . International Journal of Radiation Biology, 40, 483-492 . SCHUESSLER, H . and DAVIES, J . V ., 1983, Radiation-induced reduction reactions with bovine serum albumin . International Journal of Radiation Biology, 43, 291-301 . SCHUESSLER, H . and FREUNDL, K ., 1983, Reactions of formate and ethanol radicals with bovine serum albumin studied by electrophoresis . International Journal of Radiation Biology, 44, 17-29 .
H . and HARTMANN, H ., 1985, Chromatographic studies on the radiolysis of DNA in aqueous solution . International Journal of Radiation Biology, 47, 509-521 . SCHUESSLER, H ., MEE L . K . and ADELSTEIN, S . J ., 1976 Reaction of ethanol radicals with ribonuclease, International Journal of Radiation Biology, 30, 467-472 . SCHUESSLER, H ., GEIGER, A ., HARTMANN, H . and STEINHEBER, J ., 1987, The effect of a protein on the radiolysis of DNA studied by gel filtration . International Journal of Radiation Biology, 51, 455-466 . SCHUESSLER, H . and JUNG, E ., 1989, Protein-DNA crosslinks induced by primary and secondary radicals . International Journal of Radiation Biology, 56, 423-435 . SEDMAK, J . J . and GROSSBERG, S . E ., 1977, A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G 250 . Analytical Biochemistry, 79, 544-552 . SIMIC, M ., NETA, P. and HAYON, E ., 1969, Pulse radiolysis study of alcohols in aqueous solution . Journal of Physical Chemistry, 73, 3794-3800 . SINGH, H . and BISHOP, J ., 1984, Effect of primary and secondary radicals on chain breaks in ribosomal RNA in E. coli ribosomes. International Journal of Radiation Biology, 46, 193-199 . SINGH, H . and VADASZ, J . A ., 1983, Effect of gamma radiation on E. coli ribosomes. I . Inactivation by hydrogen radicals, hydrated electrons and secondary radicals. International Journal of Radiation Biology, 44, 601-606 . STEINMAUS, H ., ROSENTHAL, I . and ELAD, D., 1969, Photochemical and y-ray-induced reactions of purines and purine nucleosides with 2-propanol . Journal of Organic Chemistry, 91, 4921-4923 . STEINMAUS, H ., ROSENTHAL, I . and ELAD, D ., 1971, Light- and y-ray-induced reactions of purines and purine nucleosides with alcohols . Journal of Organic Chemistry, 36, 3594-3598. WADA, T., IDE, H ., NISHIMOTO, S . and KAGIYA, T., 1982, Radiation-induced reduction of thymine in aqueous solution . International Journal of Radiation Biology, 42, 215-221 . WASHINO, K. and SCHNABEL, W ., 1982, Radiation-induced molecular size changes in native and denatured deoxyribonucleic acid under anoxic conditions . Macromolecular Chemistry, 183, 697-709 . ZAREBSKA, Z . and SHUGAR, D ., 1972, Radio-products of thymine in the presence of ethanol . International Journal of Radiation Biology, 21, 101-114 . SCHUESSLER,
J.
RADIAT . BIOL .,
1992,
VOL .
62,
NO .
5, 517-526
Ethanol radical-induced protein-DNA crosslinking . A radiolysis study H . SCHUESSLER*t, G . SCHMERLER-DREMELt, J . DANZERf and E . JUNG-KORNERt
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(Received 20 March 1992; revised 9 June 1992; accepted 12 June 1992)
Abstract . Aqueous solutions of double-stranded DNA from calf thymus and bovine serum albumin (BSA) were irradiated at pH 7 under N 2 0 and N 2 in the presence of 10 -1 mol dm -3 ethanol, which was partly 14 C-labelled . Ethanol protects DNA from strand breakage by scavenging OH radicals, but ethanol radicals induce protein-DNA crosslinks . Ethanol radicals react readily with BSA mainly by addition . They react also with double-stranded DNA, but produce crosslinking only very slowly . Based on these results the following mechanism is proposed : ethanol radicals bind to BSA producing protein radicals which become crosslinked to DNA .
alteration is responsible for the loss of biological activity, further work has now been done to understand in which way secondary radicals react with DNA and proteins . Though with t-butanol radicals a higher yield of protein-DNA crosslinks is obtained than with ethanol radicals, this study has been carried out with ethanol, because 14C-labelled tbutanol was not available .
2. Materials and methods
1. Introduction Ethanol is usually considered to be a good protector against radiation-induced damage, as shown by various experiments in cells (e.g. Roots and Okada 1972, Ewing 1976) and with biopolymers (e.g. Schuessler and Hartmann 1985) . Protection is caused by ethanol scavenging OH and H radicals under the assumption that the alcohol radicals formed are inert . There are now several publications indicating a considerable reactivity of such secondary radicals with biomolecules or biological systems as ribosomes or chromatin (e.g. Nabben et al. 1983, Singh and Bishop 1983, Singh and Vadasz 1984, Herskind and Westergard 1988) . These reactions cause loss of biological activity, but the molecular mechanisms are uncertain . It was demonstrated in vitro that OH scavenging prevents strand breakage of double-stranded DNA (Washino and Schnabel 1982, Schuessler and Hartmann 1985) . However, we showed that secondary radicals formed by OH scavenging can also cause crosslinking of proteins to DNA (Schuessler and Jung 1989), although it was assumed that in cells protein-DNA crosslinks were induced by OH radicals (Oleinick et al . 1986) . Since it is possible that this *Author for correspondence . tInstitut fur Radiologie der Universitat Erlangen-Niirnberg, 8520 Erlangen, Germany . This work was supported by the Deutsche Forschungsgemeinschaft .
2.1 . Materials Highly polymerized double-stranded DNA from calf thymus (average m .w. - 2 . 107 D) was from Sigma Chemie GmbH (Munchen), 14C-ethanol from DuPont (Dreieich), lyophilized bovine serum albumin (BSA) from Boehringer GmbH (Mannheim), Sepharose from Pharmacia (Freiburg), and Aquasafe from Zinsser Analytic (Frankfurt/M .) . Other chemicals were of analytical reagent grade from Merck (Darmstadt) . Solutions were made with reagent-grade water purified by deionization, reverse osmosis, and a Milli-Q-system .
2.2 . Irradiation Radiolysis was carried out in 1 x 10 -2 moldm -3 phosphate buffer -3 )at pH 7 containing ethanol (1 x 10 -1 mol dm partly labelled with 14C . DNA (1 mg) was dis(37 MBq dm -3 =1 uCi/ml) solved in 10 ml of this solution by incubation in a refrigerator for 8 h and slow stirring for 30 min . BSA concentration was 1 .0 mg/ml . Irradiation was performed as described previously (Schuessler et al. 1987) with a Stabilipan X-ray machine (Siemens, Erlangen) with 200 kV, 20 mA, and a 2 mm aluminium filter . The dose rate was 0 .5 Gy s - 1 as estimated by Fricke dosimetry .
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2 .3 . Gel filtration Biomolecules and ethanol were separated at 4°C by gel filtration on Sepharose CL-2B (100 x 2 .5 cm) with 1 x 10 -2 mol dm -3 phosphate buffer containing 0 .02% NaN 3 at pH 7 . The eluates were collected in 3 .0 ml fractions in which the nucleic acid content was determined by the absorbance at 260 nm ; I ml per fraction was combined with 10 ml Aquasafe, and dpm were determined with a liquid scintillation counter (Primo LKB, Finland) . Protein was assayed as reported previously (Schuessler et al. 1987) with Coomassie Brilliant Blue G-250 according to the method of Sedmak and Grossberg (1977) . If BSA had to be separated from ethanol, gel filtration was carried out on Sepharose 6B (90 x 2.5 cm), and the concentration of BSA was determined by measuring the absorbance at 280 rim . Further information on the methods may be found in a thesis (Danzer, 1992) . 3. Results Previous work (Schuessler and Jung 1989) showed that reactions of secondary radicals, e .g. alcohol radicals, can cause protein-DNA crosslinks . To elucidate the mechanisms of these reactions, partly 14C-labelled ethanol was used ; DNA and BSA in a ratio of 1 :10 by mass were irradiated in the presence of I x 10 -1 mol dm -3 ethanol in 1 x 10 -2 mol dm -3 phosphate buffer pH 7 .0 under N 2 0 or N 2 . The biopolymers and ethanol were separated by conventional gel filtration on Sepharose CL-2B (Figure 1) . Highly polymerized DNA was eluted in the void volume of this column, while BSA was retarded and was found in fractions 70-100 simultaneously with ethanol . With increasing dose the amount of BSA and ethanol eluting with DNA in the void volume increased . The total amount of protein in the chromatogram was taken as 100% and the percentage of protein bound to DNA was determined . The yield of crosslinked ethanol could be measured because the ratio of 14 C- to 12 C-ethanol was known . Crosslinking of BSA and of ethanol is shown as a function of dose for radiolysis under N 2 (Figure 2) and under N 2 0 (Figure 3) . In both cases the yield of crosslinked BSA increased linearly with dose, resulting for this dose range in a constant yield of 1 . 6 ±0.3nmolJ -1 under N 2 and 0 . 7±0. l nmolJ -1 under N 2 0. Since under N 2 0 the hydrated electrons are converted to OH radicals and then to ethanol radicals, it is obvious that electrons are more effective in crosslinking BSA to DNA than ethanol radicals . This result confirms earlier findings
et al .
obtained with a nitrocellulose filter binding assay and HPLC (Schuessler and Jung 1989) . The yields of crosslinked ethanol are the same in the range of error under N 2 and N 2 0. The measured values for bound ethanol as a function of the dose follow approximately a polynomial of second order . The yields for ethanol crosslinked to DNA increase linearly with the dose (Figure 8) . To answer the question whether ethanol radicals react with double-stranded DNA, radiolysis of DNA in the presence of 1 x 10 -1 mol dm -3 ethanol was investigated . An obvious effect of ethanol is protection against double-strand breakage by scavenging all OH radicals (Figure 4) . Very high doses are necessary to show the degradation of DNA under these conditions . Since no difference was observed by changing from N 2 to N 2 0, the conclusion may be that ethanol radicals have the same low efficiency causing strand breaks as hydrated electrons . For comparison, DNA degradation by radiolysis without ethanol (Schuessler and Hartmann 1985) is shown (Figure 4) . In addition to this weak degradation ethanol radicals become crosslinked to doublestranded DNA (Figure 5) . The yield of ethanol eluted with DNA increased linearly with the dose, G=5. 2±0.8nmolJ -1 under N 2 or N 2 0. To determine how ethanol radicals react with BSA under these conditions, BSA (1 mg/ml) was irradiated in the presence of 1 x 10 -1 mol dm -3 ethanol partly labelled with 14C and analysed by gel filtration on Sepharose 6B . On this gel BSA could be separated from unbound ethanol . The monomer BSA was found in fractions 74-83, while the elution of unbound ethanol started at fraction 94 (Figure 6) . With increasing dose the amount of ethanol bound to BSA increased ; simultaneously, BSA was eluted earlier than monomet BSA . The loss of monomer BSA is not caused by unfolding of protein but by aggregation as shown by SDS-polyacrylamide gel electrophoresis in previous work (Schuessler and Freundl 1983) . The yield for the loss of monomer BSA was 2. 3 nmo1J -1 at pH 6 . 3 and 0 . 5 nmolJ -1 at pH 8 .2. The elution patterns (Figure 7) show that after 450 Gy the ratio of ethanol to BSA is about 3 :1 and is constant in the peak which indicates a rather homogeneous radiation product . With increasing dose, aggregates are formed to which more ethanol molecules are crosslinked than to the monomer BSA . The yield for crosslinking of ethanol to BSA decreased with the dose (Figure 8) . Under N 2 0 with 900 Gy, G = 97 nmol J -1 was found, but at 2400 Gy it was only 78 nmol J -1 . Under N 2 the yields were lower than under N 2 0; for _1. the same doses they were 76 and 41 nmol J A comparison of all measured yields (Figures 5 and 8) showed that the lowest was found for cross-
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Elution pattern of DNA and BSA irradiated together in the presence of 1 x 10 -1 mol dm -3 ethanol under N2 (Sepharose CL-2B) .
linking of BSA to DNA . The yields for crosslinking of ethanol to DNA and BSA to DNA were constant in the dose range up to 5000 Gy . Ethanol radicals are much more efficient in crosslinking to BSA than to DNA . At low doses under N 2 0 the yields are about 19 times higher for protein than for DNA, but they
decrease with the dose . On the other hand, crosslinking of ethanol to DNA in the presence of BSA increases with the dose and was found at 5000 Gy to be 20 nmol J -1 , which would mean that to each BSA which was crosslinked to DNA on average 30 ethanol molecules were bound .
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BSA and ethanol bound to undegraded DNA after radiolysis of BSA and DNA in the presence of 1 x 10 -1 mol dm -3 ethanol under N 2 .
rather slow . Since secondary radicals induce protein-DNA crosslinks, the assumption is that ethanol radicals add to BSA and that these protein radicals become crosslinked to DNA . Ethanol radicals are formed by reactions of OH radicals (koH •+ E1h=1 .8 x 10 9 mol -1 dm3 s-1 ) and by H radicals (kH .+Eth=1 .6 x 10 7 mol -1 dm3 s -1 ) (Dorfman and Adams 1973) . These two primary radicals produce mainly a-hydroxyalkyl radicals : 84% are a-, 13% fl- and 2 . 5% are 0 radicals (Asmus et al . 1973) . These radicals decay by association and disproportionation reactions with a second-order
4. Discussion This work confirms that ethanol protects doublestranded DNA from strand breakage . It provides further evidence that ethanol radicals are not inert but react with biopolymers and induce different modifications . Ethanol radicals react to a great extent with proteins, as shown in this and previous work (Schuessler 1975, 1981, Schuessler et al . 1976, Schuessler and Freundl 1983, Puchala and Schuessler 1986), mainly by addition . The reaction of ethanol radicals with double-stranded DNA is
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BSA and ethanol bound to undegraded DNA after radiolysis of BSA and DNA in the presence of 1 x 10 -1 mol dm -3 ethanol under N 2 0 .
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rate constant (2k=2 . 3 x 10 9 M -t s -1 ; Simic et al. 1969) . Ethanol can react with organic molecules (RH) in three ways :
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by addition R+CH 3 (~HOH-*R(CH 3 )CHOH
4.1 . Radiation-induced reactions of ethanol and BSA
(i) by electron transfer RH + CH 3 CHOH,RH' + CH 3 CHO + H + (ii) by H-abstraction RH + CH3 CHOH--•R ' + CH 3 CH 2 OH
Reaction of ethanol radicals with BSA leads to crosslinking of ethanol to BSA and to aggregation of
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Figure 5 . Crosslinking ethanol to DNA by radiolysis of DNA in the presence of 1 x 10 -1 mol dm -3 ethanol without BSA under N 2 or N 2 0 . Upper : G-values (bound molecules/ 100 eV) . (G=0. 1 molec/ 100 eV corresponds to a yield of 10 . 4nmolJ -1 .) Lower : µmoles of bound ethanol .
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Elution patterns of BSA from Sepharose 6B, before and after radiolysis in the presence of 1 x 10
the protein . At 900 Gy the yield for crosslinking under N 20 was 90nmolJ -1 (Figure 8) and under N2 70nmo1J -1 , indicating that 15 and 26%, respectively, of all ethanol radicals become bound to BSA . With increasing dose the yields decrease . A plausible explanation is that amino acid residues which react fast with ethanol radicals decrease with dose .
-1 mol
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Under comparable conditions ethanol crosslinking to two other proteins have been measured . With ribonuclease the yield for crosslinking under N 20 with 5kGy is 60 and under N 2 50nmolJ -1 (Schuessler 1981) . With haemoglobin the yields are similar, 54 under N 20 and 48nmolJ -1 under N 2 (Puchala and Schuessler 1986), but constant up to a dose of 4 .8 kGy .
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Elution profile of BSA and the ratio of bound ethanol molecules per BSA molecule after radiolysis of BSA in the presence of 1 x 10 -1 moldm -3 ethanol under N2 0 -
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dose (Gy) G-values (bound molecules/ IOOeV) for crosslinking of ethanol, BSA, and DNA after irradiation under N 2 0 at pH 7 . = crosslinking between ethanol and BSA in the presence of DNA ; O = crosslinking between ethanol and DNA in the presence of BSA ; O =crosslinking between BSA and DNA in the presence of ethanol. (G=1 molec/100 eV corresponds to a yield of 104 nmol J -1 . )
Figure 8 .
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Pulse radiolysis studies (Schuessler and Davies 1983) of secondary radicals and BSA gave evidence that formate radicals with a redox potential E°(C0 2/CO2 ) = -2 . 0 V can transfer electrons to the disulfide groups in BSA . In contrast, ethanol radicals with a redox potential E°(Eth/Eth')= -1 .2 V (Breitenkamp et al . 1976) have a low rate and yield for electron transfer, so that with 1 .5 x 10 - 5 mol dm - 3 BSA, the concentration used in this work, only 1-2% can react in this way . This result was also confirmed by studying radiation products after steady state radiolysis of BSA and secondary radicals (Schuessler and Freundl 1983) . While the reaction of formate radicals yields aggregates of BSA by the formation of intermolecular S-Sbridges, the reaction of ethanol radicals leads only to non-reducible aggregates, indicating that ethanol radicals did not transfer electrons to cystine in BSA . It was previously assumed that aggregation can be induced by H-abstraction by ethanol radicals : CH3HOH + ProtH-+CH 3CH2OH + Prof Prof + Prot'--- Prot - Prot Prot' + Prot-+Prot - Prof However, our experimental results show that crosslinking of ethanol to BSA is more efficient than aggregation. Also it is obvious that more ethanol molecules per BSA molecule are bound to BSA aggregates than to monomer BSA (Figure 7) . For these reasons it is now assumed that the main reaction under N 20 is the addition of ethanol radicals to BSA molecules . CH3HOH+Prot-*(Prot-CHOH)' CH3 The protein radicals can react further with ethanol radicals or can produce aggregation . In accordance with all studied proteins a higher percentage of ethanol become bound to BSA under N 2 than under N20. A plausible explanation is that hydrated electrons form BSA radicals which then react with ethanol radicals : eay + Prot-+Pros' CH 3 CHOH + Prof -- Prot - CHOH CH3 4.2. Radiation-induced reactions of ethanol and DNA In this work crosslinking of ethanol to doublestranded DNA could be detected after radiolysis
under anaerobic conditions . The yield under N 2 0 as well as under N 2 is 5nmolJ -1 (Figure 5), which means that only 0 .8 and 1 .5%, respectively, of all ethanol radicals become crosslinked . It is known that alcohol radicals react with nucleobases. Addition products between ethanol and thymine as well as with dimethylthymine and thymidine have been found (Brown et al . 1966, Zarebska and Shugar 1972, Cadet et al . 1981) . In more recent work (Schuchmann et al . 1986) reactions of methanol radicals with dimethyluracil and dimethylthymine have been studied . The analysis of the radiation products show that the hydroxyalkyl radicals add with a very high specificity to the C(6) position with a yield of 80 nmol J -1 at a dose rate of 0. 19 Gy s -1 . This yield can be increased by reducing the dose rate, because the rate constant for hydroxymethyl radicals with pyrimidine is only about 1 x 104 mo1 -1 dm3 s -1 . While about two-thirds of the hydroxymethyl radicals add to dimethyl thymine, one-third abstracts an H-atom from the methyl group . Both thymidine radicals can react further with a second ethanol radical or undergo dimerization . The a-hydroxyalkyl radicals also react with purines forming compounds alkylated at C(8) (Steinmaus et al. 1969, 1971) . Single-stranded DNA from 0 X 174 can be inactivated by iso-propanol radicals (Nabben et al . 1983) . For this reaction a rate constant of 7.4 x 10 3 mol -1 dm3 s-1 was measured . Experiments with light-induced reaction of a-hydroxyalkyl radicals with single-stranded DNA showed that the rate is an order of magnitude slower than with nucleobases and an additional order of magnitude lower with double-stranded DNA (Livneh et al. 1982) . To our knowledge there is no publication on irradiation-induced reactions of ethanol with double-stranded DNA . The low yield for crosslinking of ethanol to DNA, which was found in this work, is consistent with data from the relevant literature . Accordingly, it is assumed that crosslinking is caused by addition and H-abstraction . Since the yield under N 2 0 and N 2 is the same, DNA radicals produced by hydrated electrons seem to react also with ethanol radicals . Furthermore, it was observed that ethanol radicals and ethanol radicals with hydrated electrons cause a slight degradation of DNA observable only with very high doses (Figure 4) and almost negligible in comparison with degradation caused by OH radicals . Strand breaking of DNA by ethanol radicals may be induced by Habstraction from DNA .
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Ethanol radicals with BSA and DNA 4 .3 . Radiation-induced reactions of ethanol with BSA and
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DNA
The main reason for this work was to investigate the induction of protein-DNA crosslinks by secondary radicals . Under conditions of complete scavenging of OH radicals by ethanol, the yield for crosslinking BSA to DNA is still 1-5 under N 2 and 0. 7nmolJ -I under N 2 0 (Figure 8) . These values are similar to those published previously which were 1 . 5 and 0.4nmo1J -I , respectively (Schuessler and Jung 1989) . Simultaneously, with BSA, ethanol molecules become bound to DNA, in fact rather efficiently, increasing with dose from 9 to 30 ethanol per BSA . Furthermore, no aggregation of BSA could be found under this condition . The prevention of aggregation of BSA by the presence of DNA has previously been observed, when crosslinks were induced by primary radicals. It was assumed that there is an electrostatic interaction between DNA and BSA which contributes to the yield of crosslinks . This assumption could be confirmed by lowering the pH which leads to stronger interaction and a higher yield of crosslinks between BSA and DNA (Schuessler et al . 1987) . If the ratio of BSA : DNA is 10 :1 by mass and the dose 440 Gy under N 2 , there are 1500 BSA molecules crosslinked to one DNA molecule with a molecular weight of 2. 107 D . Considering the dimensions of the two biopolymers, between 700 and 2500 BSA molecules can become attached on the two sides of one DNA molecule . When the ratio BSA :DNA is increased over 10 :1 by mass, then the yield of BSA-DNA crosslinks decreases and aggregations of BSA occurs again (Goetzinger 1992) . This work shows that the efficiency of ethanol radicals is much higher in adding to BSA than to DNA. Therefore, if BSA and DNA with a ratio 10 :1 are in solution, ethanol radicals will attack the protein exclusively, producing protein radicals, mainly by addition and H-abstraction . These protein radicals react further with other ethanol radicals or with DNA, but not with another BSA molecule because they are not free to diffuse . If BSA alone was irradiated with ethanol under N 2 0 with a dose of 2400 Gy, the ratio of ethanol crosslinked to BSA is 12.5 :1 on average, while with the same dose this ratio is 24 :1, if BSA is bound to DNA . This high binding ratio of ethanol to BSA reflects the lower chance of the BSA radical participating in a crosslinking reaction to DNA than to an additional ethanol radical . This is similar to the aggregation of proteins induced by ethanol radicals . Aggregates also have a higher amount of crosslinked ethanol than the separated monomer (Figure 7) .
Under N 2 the yield for crosslinking BSA to DNA is twice as great as under N 2 0 . This is because the hydrated electrons are more efficient in producing protein radicals to crosslink with DNA, but the ratio Eth :BSA is lower than under N 2 0. At 2400 Gy this ratio is 8, while if radiolysis is carried out in the absence of DNA it is about 7 . This study indicates that ethanol radicals add to proteins in preference to DNA, forming protein radicals which become crosslinked to DNA . Accordingly, it has to be assumed that in cells the primary target by secondary radicals is not DNA but more probably proteins, and that besides strand breaks protein-DNA crosslinks may cause loss of biological activity . Furthermore, this work illustrates how complex radiation-induced reactions between biopolymers in a solution become with the addition of only one substance and that further investigations are required to understand the molecular radiation biology of the cell .
Acknowledgements The authors thank the Deutsche Forschungsgemeinschaft for financial support of this work .
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H . and HARTMANN, H ., 1985, Chromatographic studies on the radiolysis of DNA in aqueous solution . International Journal of Radiation Biology, 47, 509-521 . SCHUESSLER, H ., MEE L . K . and ADELSTEIN, S . J ., 1976 Reaction of ethanol radicals with ribonuclease, International Journal of Radiation Biology, 30, 467-472 . SCHUESSLER, H ., GEIGER, A ., HARTMANN, H . and STEINHEBER, J ., 1987, The effect of a protein on the radiolysis of DNA studied by gel filtration . International Journal of Radiation Biology, 51, 455-466 . SCHUESSLER, H . and JUNG, E ., 1989, Protein-DNA crosslinks induced by primary and secondary radicals . International Journal of Radiation Biology, 56, 423-435 . SEDMAK, J . J . and GROSSBERG, S . E ., 1977, A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G 250 . Analytical Biochemistry, 79, 544-552 . SIMIC, M ., NETA, P. and HAYON, E ., 1969, Pulse radiolysis study of alcohols in aqueous solution . Journal of Physical Chemistry, 73, 3794-3800 . SINGH, H . and BISHOP, J ., 1984, Effect of primary and secondary radicals on chain breaks in ribosomal RNA in E. coli ribosomes. International Journal of Radiation Biology, 46, 193-199 . SINGH, H . and VADASZ, J . A ., 1983, Effect of gamma radiation on E. coli ribosomes. I . Inactivation by hydrogen radicals, hydrated electrons and secondary radicals. International Journal of Radiation Biology, 44, 601-606 . STEINMAUS, H ., ROSENTHAL, I . and ELAD, D., 1969, Photochemical and y-ray-induced reactions of purines and purine nucleosides with 2-propanol . Journal of Organic Chemistry, 91, 4921-4923 . STEINMAUS, H ., ROSENTHAL, I . and ELAD, D ., 1971, Light- and y-ray-induced reactions of purines and purine nucleosides with alcohols . Journal of Organic Chemistry, 36, 3594-3598. WADA, T., IDE, H ., NISHIMOTO, S . and KAGIYA, T., 1982, Radiation-induced reduction of thymine in aqueous solution . International Journal of Radiation Biology, 42, 215-221 . WASHINO, K. and SCHNABEL, W ., 1982, Radiation-induced molecular size changes in native and denatured deoxyribonucleic acid under anoxic conditions . Macromolecular Chemistry, 183, 697-709 . ZAREBSKA, Z . and SHUGAR, D ., 1972, Radio-products of thymine in the presence of ethanol . International Journal of Radiation Biology, 21, 101-114 . SCHUESSLER,
