Radiolytic damage to genetic material - Journal of Chemical Education

Radiolytic damage to genetic material. John F. Ward. J. Chem. Educ. , 1981, 58 (2), p 135. DOI: 10.1021/ed058p135. Publication Date: February 1981...
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Radiolytic Damage to Genetic Material John F. Ward Division Of Radiobiology, DqmtmMof Radiology. School of Medicine, W 2 7 . University of Califmia. San Diego, La Jolla, CA 92093

The radiation chemistry of "genetic material" is of interest not only because of several unique facets of the chemistry involved (particularly as a long chain polymer) but also because of its biological significance. In studies of the radiohiologv of bacterial and mammalian cell systems it has been quite"hell estahlishtd t h . ~ tthe rrliulnr genome is the most significant site ior r~dinticmi n d ~ ~ c ck di i i ~ n rand murageneais. he many lines of evidence supporting this conclusion have been summarized by Hutchinson ( 1 ) and more recently by Painter (2).Thus studies of radiation damage to DNA receive impetus from (a) current concerns of the possible hazards of exposure to low levels of radiation and (b) attempts to devise ways of improving the efficacy of radiation therapy in sterilizing cancers. We are fortunate, therefore, that a broad hase of knowledge has been ---. ...- develoned in the radiation chemistrv " of genetic material. 0hvio&ly cellular complexity prevents a direct attack on the nroblem so that the maioritv . . of our understand in^ comes from.studies of model systems of nucleic acid constic uents irradiated individually. One of the major objections raised by biologists about such studies is that the doses of radiation used (kilo rads-mega rads) are far greater than those used with mammalian cells (100-1,000 rads). In response it should be pointed out that the percentage of molecules sur\.iving in the typicnl ratl~,itic,nchemirtr) experiment i> most often greater th.in tht. Iwrcrlttagt of cells survirine in a biology exp&ment. In this discussion some of the basic findings in the radiation chemistry of genetic material derived from studies of model systems will be described. Then an attempt will be made to use the findings to extrapolate to the consequences of radiation damage to DNA within cells. ~

~~~

~

~

shows schematically the backbone while Figure 1B shows the molecular structures involved in the repeating deoxyribose phosphate structure. Attached to the 1' position of each de-

-

Structure

The DNA molecule consists of a backbone of deoxyribose moieties linked together hy phosphodiester bonds. Figure 1A

Figure 1. Schematic repesentations of DNA sbudure: A. Backbone. 8. Bonds involved in backbone.

&" H Thymine

rC

Cytosine

:igure 2. DNA base structure.

Volume 58

Number 2

February 1981

135

mini is necessary before reconstruction of the native structure, it is imperative that the chemical identities be known.

Hydroxymethyl Uracil

Mechanisms of Radiation Damage . In order to assign chemical structures to radiation damaged sites in genetic material it is important to use model svstems. ~ r o d u c i i o nof sufficient amo&ts of damage for structure analysis in genetic material irradiated intracellularly would require large, non-biological radiation doses. In order to select a model system for study which is valid to the in uiuo situation. it is imoortant to know which orocesses of damage production occur within the living cell. In recent vears several lines of evidence sueeest that the hvdroxvl . radicai.OH produced by radiolysis of water is involved in approximately 70% of cell killing (9-11 ). In these experiments compounds capable of scavenging hydroxyl radicals were added to cells prior to radiation. Thus it was found that cell radiosensitivity could be reduced by a factor of 3 a t high concentrations of scavenger. The ahility of a compound to protect (measured by the reciprocal of the molarity needed to accomplish 50%of the radioprotection possible) is directly proportional to the absolute rate constant for its reaction with -OH radicals. Clearly studies of reaction of this reactive species with DNA constituents is an ideal model for 70% of the lethal damage. The amount of cell killing which is not caused hv .OH radicals is not related to the reactions tJ'thc hydrared electron (91and is more likely due to dirrct i.,niz;lti.m ..I the molecules r : direct ioni n \ h e d . Hence studies o f d a n ~ i ~ri tvw l t ~ t ~trom izatiun of DNr\ constituent: I n n i r n w d t m d e l fcr rhe residual 30% damage. Direct lonizalion of Nucleic Acid Constituents The most widely used method for observing the effects of ionizing radiation on pure solid materials is electron paramagnetic resonance spectroscopy, EPR. The principles, possibilities, and problems of this technique have been covered by Dr. M. Sevilla a t this symposium (12). The major initial reaction of ionizing radiation with molecules is ionization, and in m a w instances the initial cation rndical cnn he observed (13).It is importml to realize that the f s extraooinitial radical is the species w e wiqh t o ~ d c n t ~ior lation to biological systems. The reason is that the subsequent reactions of the initial species after their formation depend to a large extent on their immediate environment. In studies of pure compounds the reactions of initial species (the pristine cations (13)) will he with parent molecules hut within the biological system it will react with a heterogeneous environment of molecules including water. In order to accomplish the identification of the initial cation EPR spectroscopists work a t very low temperatures and very early times post-irradiation i.e., prior to subsequent radical reactions. The s cation radicals formed on irradiation of DNA hases are thought to be the precursors of most other oxidation radicals observed. In general the loss of an electron from a DNA hase to form a cation does not occur from any particular site in the molecule hence the term a cation. T h e r cation, however, will react further to give a defined free radical usually uncharged (eqn. (1)).

-

8 Hydroxyadenine Figure 3.Radiation products of DNA bases which retain planar structure: A. Hydroxymethyluracil B. 8. Hydroxyadenine.

oxyribose is a heterocyclic hase, the four hases involved in DNA are shown in Figure 2. The glycosylic bond is with the backbone structures running in opposite directions, the structure being stabilized by hydrogen bonds between comolementarv bases. The amount of such DNA in the 46 chromosomes df a human cell is -6 X 10-'2g-about 2 m in length per cell. Thus the DNA must be quite tightly packed and consequently any studies of radiation chemistiy of isolated DNA must be considered with this thought in mind. Types of Damage Ionizing radiation is known to cause alterations to both hase and backbone, Base damaee can be of two tvDes: either. (a) saturation of the ring struckre causing loss o{&a&ity of the hase. loss of stacking" interaction with neiehhorine bases and hence physical distortion of the molecule, or (b) reaction with exocyclic groups leaving the planarity of the ring intact, e.g., formation of hydroxymethyl uracil from thymine (Fig. 3A) or 8 hydroxy adenine from adenine (Fig. 3B). Damage of type (a) may be detected easily by cellular repair systems while type (b) because of lack of . ohvsical distortion mav not be recoe" " nized and hence not repaired. Few studies of hase damage in uiuo have been carried out and in " eeneral are limited to thvmine damage measurement by the procedure developed by Hariharan and Cerutti (3). Most biological studies have concentrated on single strand breaks probably caused hv damage to deoxvribose moieties. Thead;antared ~hesest&Iiesis;heavailability of sensirive assay procedures ( . I t i ) which ran he used in the hioloricnllv significant dose range. More recently an assay procedure fo; double strand breaks has been developed (7). No doubt, this will attain wide usage since the DNA double strand break is thought to be a lethal event within a cell. The fourth type of damage which has not received a great deal of attention is the formation of DNA-protein cross-links (8).The absence of sensitive assavorocedures has limited such -. measurements in uiuo. Within the cell all of these tvoes of damaee are suhiect to the cell's attempt to repair 01" iemove the ;amage. eonseauentlv enzvme svstems are to he assiened to the - if specific . repair process, it is necessary to know the chemicil identities of the various types of damage. For instance, the term strand break may he physically descriptive of the event which occurs, but it gives us no information about the termini a t either side of the strand break. Since enzymatic processing of these ter136

Journal of Chemical Education

:,

RHt-R'+Hi

(1)

Thus for the cation radical of thymine the proton is lost from the exocyclic methyl group (141, cytosine from the one position (15) while guanine and adenine can deprotonate from several positions (13). Of course the subsequent reactions occurring with any radical depend upon its immediate environment, in the pure material only reactions with similar molecules can occur. In another environment (e.g. aqueous) different reactions are possible. Sevilla (16)has shown that r cations, produced in alkaline aqueous glasses by photolysis, can react with hydroxyl ions from the matrix giving an 'OH radical addition product (eqn. (2)).

.

It will he seen below that these two neutral radical products of s cations (eqns. (1)and (2)) are closely related if not identical to those formed by .OH radicals with the same parent compounds. It is not possihle a t this time to review the field of EPR soectroscoov of irradiated DNA constituents. However. the significancebf the area in radiobiology should he clearl;recoenized. The imnortant anestions vet to be answered in this aren art.: ( 1 ) What are the rclutiv~.yieldsof the different radicals ior~ncduoon irradiuticm of DNA constittirnts? 121 What are the expected suhiequenr reo~.tionsofthe prt.tiuecations u,irh a 4niulated cellulnrenrir.~timentQ(31 What are the relative rates of the various possible subsequent reactions? I t will be noted that nothing has been said about reactions of the reduced radicals (anion radicals). While the literature is replete with such studies, it is the author's choice not to attempt to review the field. If a justification for this prejudice is required-the reducing species formed in water radiolysis e-., is apparently not significant in causing cell death (9). The intracellular source of e-,, is not necessarily water, and by the same token the source of the reduced organic species is not necessarily another organic molecule. The adducts of electron reactions, he they of hydrated origin or not, with organic molecules would he expected to he similar. It should he emphasized that the problems inherent in tracing a molecular mechanism from a cation radical parent to a final stable radiation product are exceedingly complex. In contrast it is feasible to trace the course of'OH radical reactions using the techniques described in previous papers presented in this symposium.

'l'hus the rate cmstants for re:~ctionk l can III. determined (if that l h w ;m(l .SI. do not c h . t n- s durina- the i t , . .~..urned . .. course of reaction. Typical rate constants for reaction are shown in the table. The major reaction of .OH appears to he with the double bonds of the molecules (5,6 for pyrimidines and 4,5 for purines). For instance the reaction of 'OH with thymine is almost exclusively with the 5,6 double hond, this has been shown by pulse radiolysis linked up to EPR (20) and with chemically produced .OH radicals (21). In the case of thymine approximately equal amounts of addition occur at C g &d Cg, kt with cytosine addition is almost exclusively a t C5(determined by a ~ u l s conductometric e techniaue) . . (22) . . The only exceptions to addition reactions so far recorded are 'OH ahstraction of a hydrogen atom from the exocyclic methyl group of thymine (limited to about 3% of the total reaction a t neutral DH (23))and ahstraction of the 8 hvdroeen , .. frwn purtnes i241. \\'ill>vn6.r n l (2,5I suggest tli;st .OH radicals rtnc r aith guanine deriwtlrei hy electron traoiter reaction (511.

To use thymine as a typical hase, and indeed most work has been done with this hase. the suhseauent reactions of the hydroxyl radical adduct 'can he des'crihed. This reaction scheme (6) was orooosed in 1960 to account for the hieh vield of thymink hyd;oxy hydroperoxide (26) (only reactiois df the 5,6 double hond are shown).

Hydroxyl Radical Reactions with DNA Constituents Base Damage The DNA hases (Fig. 2) react a t almost diffusion controlled rates with 'OH produced by water radioloysis. These rates have been measured by various pulse radiolysis techniques, either (a) directly by measuring the rate of production of the hase OH. transient (171,(b) by measuring the rate of removal of the UV absorbance of the parent molecule (18). or (c) by competing the hase against a standard reactant for the 'OH (19) radical. Thus in the reaction scheme below: Base + .OH % ~ a s e - OH.

(3)

In (a) d(Base - .OH)ldt is measured = kl[Base][.OH] (h) (d BaseVdt ismeasured = -kl[Base][OH] and (c) the amount of SOH. formed in the presence (SOH,) and in the absence (SOH,) of hase is measured.

'OH Rate Constants Reaction 'OH 'OH 'OH 'OH

+ 'OH + e.,

+ Base + Deaxyribase In nucleotidee

Rate Constant mole-'sec-' 5 X 109 3 X 10'O 4 X lo9

0.8 X

10'

'Assumes lhat redunion In base damags p i n g horn base M nuclearide is due to Icausnging 0f'OH by dsoxyribooe.

The thymine 'OH adduct treaction 6, is still n mdical and r t w t i nipidly with other radicals (reacrion8) in this L.ase with m o l ~ ~ c uoxygen lu dissolved in rhe aqueous s~,luIim(saturated 0 2 solurion at 2j°C is 1.3 mM,. This is a well ~ r n h l i s h e dreaction of oxygen which in ib nound smte is n l~i-mdiral.Again the radical formed is reactive and can be reduced hy reaction with a reducmg species such as 0 2 - ion radical (reaction 9). fornled by reaction of tht. hvdrared electron with molecular oxygen dissolved in the aqueous solution. tln nwtion t7j the reducing species is designated H',H02'sirre i n IYtiO the h) drated electron had nut hrtw disc overed.^ 'l'hr I onfirmation of the various steps of this mechanism by modern techniques is described in reference (26). Since the proposal of this basic mechanism a large variety of thvrnine radiation oroducts has been found includine 8 hydroperoxides (28). f'hese probably arise from the varigus pathways available a t each step of the reaction scheme which include (a) site and stereochemistry of 'OH attack (b) stereochemistry of 0 2 addition, alternate reactions with other radicals, (c) alternate reactions with other radicals, and finally breakdown of overall products. However, the majority . . of the producu are of the rink d;~magedtype, and in severol instances fragmentation of the ring uccurs. While the reaction schemes forthe other bases is notknown as clearly as that for thymine somewhat similar mechanisms can he predicted. The 'OH adducts of cvtosine and adenine react raoidlv with oxveen hut that of guaiine (using guanosine as a &d& cannot 6e seen to react usine the nuke radiolvsis techniaue . (29). . . The hydro;!, hydn8pntxidr;,fcytosine isstahlt. only in acid solution and consequently cannot be isolated in neutral (26). Volume 58

Number 2

February 1981

137

Several other radiation products of cytosine have been described. The radiation chemistry of the purines in aqueous solution is a problem mechanistically since in contrast to the pyrimidines only a fraction of the initial hydroxyl radical attack shows up as destruction of the chromophore. It is believed (26) that hack reactions among the various radicals causes reformation of the parent molecules together with the damaged hase products isolated. Thus in the nreseuce of oxveen the maior . .oroducts of the DK.4 hasra 1;1rn1~d s~lhrt.quenrlo .OH radical auack Ii&e I t s t their U\' a h i o r p t ~ mFor tllc pyrimidines ever! ,011 nrt.18 k leads to deitructiull c , i c