Biochimica et Biophysica Acta, 1071 (1991) 103-122 © 1991 Elsevier Science Publishers B.V. 0304-4157/91/$03.50 ADONIS 030441579100055H

103

Review

BBAREV 85380

The effect of ionizing radiation on lipid membranes G. Stark Department of BioloD', University of Konstanz, Konstanz (Germany) (Received 6 September 1990)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103

II. The chemical basis of the radiation effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Direct versus indirect radiation effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The radiation chemistry of water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Important secondary radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The inverse dose rate effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

104 104 104 105 106 107

I II. Structural consequences of lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111

IV. Ion transport through planar lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The experimental set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The effect of lipid peroxidation on K +-transport mediated by macrocyclic ion carriers . . . . . . . . C. The inactivation of an ion channel: gramicidin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 113 114 116

V.

120

Biological implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121

I. Introduction The molecular basis of membrane biology has greatly profited by the study of lipid model systems. Suspensions of liposomes, planar bimolecular lipid membranes as well as lipid monolayers have contributed to clarify the structural organization of biomembranes and the mechanisms of membrane transport. The biological background of the present article is represented by the field of cellular radiobiology. The targets of radiation action inside the cell, the mechanisms of cell killing, of radiation induced mutagenesis and of cancerogenesis are still a matter of considerable controversy. Though the genetic substance is generally believed to represent the most important radiation target, the role of other cellular constituents can certainly not be neglected. This holds especially for the great variety of cellular membranes. Their importance, apart from many other cellular functions, might also

Correspondence: G. Stark, Fakulfiit fiir Biologie, Universit~it Konstanz, Postfach 5560, D-7750 Konstanz, Germany.

extend to the biological functions of the genetic substance. The close adherence of DNA to the nuclear membrane (in eukaryotic cells), at least in principle, allows a transfer of a radiation induced membrane damage to the level of the genetic substance [1]. The radiobiology of membranes has been summarized repeatedly [2-5]. Contrary to previous reviews, which provide a general survey about radiation effects on biomembranes, the present article concentrates on the lipid component. Two main objectives are envisaged: Radiation induced modification of membrane transport and the 'inverse dose rate effect'. Based on studies with model systems, mechanisms are discussed, by which free radicals, generated by absorption of radiation, interact with lipid membranes and modify their transport properties. The article concentrates on ion transport through lipid membranes in the presence of model compounds, the structure and function of which has been investigated in considerable detail. The study of the so.called "ionophores', i.e., of ion carriers and of channel forming substances in lipid membranes, has provided information about the fundamental mechanisms of ion movement across biological

104 membranes. The author's interests throughout recent years has been devoted to the question, how the action of these model compounds is modified by exposure to ionizing radiation or to ultraviolet-fight. Studies of this kind represent a first step towards the clarification of radiation action on more complex transport phenomena of biological membranes. The transport systems under study have been found to be influenced by radiation in two di[ierent ways: They may either be inactivated by fre~ radicals (e.g., the gramicidin channel) or they respond to radiation induced lipid peroxidation. In the latter case they may be used as sensors of a radiation induced structural modification of the membrane (e.g., the ion carriers of the valinomycin type). Lipid peroxidation has been found to show a so called inverse dose rate effect, i.e., the effect, at constant radiation dose, increases with decreasing dose rate. This is contrary to many other cellular radiation effects, which show a 'normal dose rate effect', i.e., a decreasing effect at increasing dose rate. Products of lipid peroxidation have been suggested to interact with DNA and to contribute to mutagenesis and to cancerogenesis. Therefore, the inverse dose rate effect observed at radiation induced lipid peroxidation might have considerable consequences for the evaluation of the genetic risk and of the cancer risk following radiation exposure of man. The article summarizes the experimental basis and the interpretation of the inverse dose rate effect. The effect is a direct consequence of a radical chain mechanism well-known in polymer chemistry. I!. The chemical basis of the radiation effects

Gray (1 Gy = 1 J/kg) to about 10 3 Gy. The direct radiation effect may be estimated on the basis of the hit theory [6]: The radiation dose D37 necessary to modify 6370 of molecules of mass /~ by a single hit (in an arbitrary part of the molecule) is D37(direct ) =

(1)

W H/~t

(Wu = mean hit energy). Assuming a molar mass M = L# ( L - Avogadro's constant) of 102-104 g/mol (this is the range of values encountered with lipid molecules and with single transport units discussed in the present article), and assuming WH = 60 eV, one obtains D37 (direct) approx 6. 107-6 • 105 Gy. The values are several orders of magnitude larger than those found experimentally. This indicates the importance of the indirect radiation effect initiated by the freely diffusible radicals of water radiolysis. Further support for the indirect effect is obtained from experiments with radical scavengers (see below). H-B. The radiation chemistry of water The chemical processes following exposure of water to ionizing radiation have been described in many review articles and textbooks. Refs. 7 and 8 contain a highly readable compilation of previous and more recent developements. Fig. 1 shows a simplified scheme of reactions representing the main aspects of water radiolysis. Absorption of radiation leads - via ionization and excitation of water molecules - to the primary water radicals O H . , H , and the hydrated electron e~q. The fourth radical, HEO+, reacts within less than 10 -[4 s to O H - and to H30 +.

H-A. Direct versus indirect radiation effect The lipid component of biological membranes corresponds to a bimolecular structure with a thickness of about 5 nm, which is surrounded on both sides by aqueous phases. The thickness of (at least one of) the aqueous layers is many orders of magnitude larger as compared to the membrane thickness. This holds for the cytoplasmic membrane as well as for the great variety of membranes surrounding the intracellular organelles. Therefore, free radicals generated by water radiolysis (see below) have uclimited access to attacking and to chemically modifying the membrane constituents. This is usually designated as an indirect radiation effect to be distinguished from the direct radiation effect, where radiation is absorbed directly by the molecule considered. A simple estimate indicates that (at least for artificial lipid membranes) the indirect radiation action is by far more important compared with the direct radiation action. The effects treated in this article were observed at radiation doses ranging from small fractions of a

H20 excltotSon

o

H20 ~"

n

H20 *

_

/ dissociation

1 L,.. o,.

1

Padicll track

÷ n H20

+ H20

,

reactions of

Sonizing

t|ong

the]

Par~ic|es

1 OH; H; e,~q, H2, HsO*, OH-, H2O2 (products of Nater radiolgsis) Fig. 1. Reaction scheme of water radiolysis.

105 TABLE I

G.values of the products of water radiolysis following exposition of deoxygenated neutral water to radiation of low linear enerD, transfer (e.g. y-radiation or fast electrons) [7,8]. Species:

OH-

H.

eaq

H+

OH-

H2

H202

G-value:

2.7

0.55

2.65

3.3

0.6

0.45

0.7

The concentration of radicals is f~irly high along the tracks of ionizing particles in water, made up by clusters of ions (spurs). The latter are formed either by primary ionizing particles used for water irradiation (fast electrons or a-particles from an accelerator or a radioactive nuclide) or - if water is exposed to energetic photons (X-rays or v-rays) - by secondary electrons liberated by the absorption of the photons (via photoelectric effect, Compton effect or pair production). The initial radical concentration, due to its high local concentration along the track, is reduced considerably by secondary radicalradical reactions, such as H ° + H ° ---,H 2. Their rate constants are of the order of 101° M-1 s-1, i.e., more or less diffusion controlled. By way of these reactions and by way of free diffusion, the inhomogeneous distribution of radicals, after about 1 0 - 7 S, is converted into a fairly homogeneous concentration of much smaller amplitude. Table I summarizes the G-values of the various products. This quantity has been introduced to characterize the concentration of the various products in water after the completion of the intra-track-reactions. It is defined as the ratio: number of molecules of a given product species per radiation energy absorbed. The latter is usually expressed in units of 106 eV. Consequently, the aqueous concentration of a species with G = 1, after a radiation pulse of 1 Gy = 1 J / k g absorbed dose, corresponds to 1.03.10 - 7 M.

H-C. Important secondary radicals Primary water radicals undergo chemical reaction with many solutes found in aqueous solutions of biological relevance. In this way a great variety of secondary radicals are formed which on their part may also show high reactivity towards other substances. This shall be illustrated by two important examples: In the presence of oxygen, the concentrations of H ° and of ea~, via reactions (2) and (3), are strongly reduced in favour of the radical species 0 2 " and HOE" : eaq + 0 2 ~ 0 2 "

(2)

H.

(3)

+ 0 2 -4

HO~"

The superoxide radiea| 0 2 • ""d the .,,.-h,.a . . . . . 1 radical HO 2 • are in a pH-dependent equilibrium (4): HO 2" ~ 0 2 • + H +

(4)

The pK a of the weak acid HO2- is 4.8 [9], so that at pH > 4.8 the superoxide radical is predominant. The concentrations of H ° and ea~, because of reactions (2) and (3), virtually disappear in O2-saturated water. Therefore, the G-value for the conjugated pair HO 2 • / O 2 ° corresponds to the sum G(H ° ) + G(ea~) in the absence of oxygen (i.e., G(HO 2 •/O2

° ) = 3.2).

The reactivity of the oxygen radicals towards compounds of biological interest have been studied in great detail (see Refs. 8 and 9 for a review). The superoxide radical has been suggested to represent a major factor of oxygen toxicity in biological systems (c.f.e.g., Ref. 10 for a discussion of this controversial problem). The OH • radical does not react with 02. It is the main oxidizing radical that is formed when aqueous solutions are exposed to radiation. The main types of reactions undergone by OH • may be classified as follows: (a) electron transfer reactions with inorganic and organic compounds (e.g., the formation of CI 5 • radicals, see below), (b) abstractions of carbon-bound Hatoms (see initiation of lipid peroxidation), (c) additions to double bonds (e.g., to the indole ring of tryptophan, see inactivation of the gramicidin channel). The OH • radical is able to form secondary radicals of high reactivity. This is illustrated with CI 5 ° This radical is spectrophotometrically detected, if CI- conraining aqueous solutions are irradiated [11-13]. In view of the comparatively large cytoplasmic CI- concentration of > 0.1 M, the CI 2 • radical may be assumed to play a role as an intermediate radical species in the developement of OH o initiated cellular radiation damage. The following scheme of reactions has been proposed [13]: OH- + C I - # C I O H - -

(5)

CIOH-" + H ÷ # C I - + H 2 0

(6)

Ci. + C I - ~ CI [ •

(7)

Alternatively, instead of reactions (5) and (6), formation of CI- by reaction of CI- with H zO+ in the track of ionizing particles has been suggested [12]. The rate constants of CI 2 • induced chemical reactions have been found to be about 1-2 orders magnitude smaller compared with the primary OH • radical [14,151. While the 0 2 " radical shows comparatively high reactivity towards compounds of cellular origin, there is another class of OH ° initiated reactions leading to secondary radicals of low reactivity. The corresponding

106 substances may be used as radical scavengers. To distinguish between the effect of the O H . radical and the effect of other radical species, experiments may be performed in the presence of alcohols, such as t-butanol. By way of the reaction (8)

OH- + C(CH3)3OH -~" CH2C(CH3)2 OH + H20

the highly reactive OH- radical is converted into an alcohol radical of considerably smaller reactivity. There are two types of radicals which determine the radiation chemistry of air saturated water at sufficiently low pH: The radicals OH ° and HOe ". Their relative contribution to a radiation effect observed may be estimated by performing experiments in the presence of formate. Through reactions (9) and (10) OH.

+HCOOH

-~ . C O O H

+ H20

(9)

.COOH+ O2 --, C02 + H02.

(10)

the O H . radical' is transformed into a perhydroxyl radical, i.e., the effect of the latter may be studied without the interference of other radicals.

H-D. Lipid peroxidation This term is commonly used for the reaction of lipids (LH) with molecular oxygen to form lipid hydroperoxides (LOOH) according to the general formula LH+O2 --, LOOH.

(11)

The reaction, however, does not proceed as suggested by eqn. 11. Because of the electronic configuration of oxygen and of lipids, namely triplet versus singlet, the uncatalyzed lipid peroxidation is spin-forbidden [16,17]. Spin restrictions are removed, however, if lipid per-

CHs'CHa'CH"CH'CHa'CH'-CH'CHa'CH'CH"(CH~)r'COOH

(LH)

,[ ~ H#

N

CHs'CH2"CH-'CH'C.H'CH:CH'CH2"CH'--CH"(CHa)T -COOH D|ene

(L')

CHs'CH2"~H'CH=CH'CH=CH'CH2"CH:CH'(CH2)T-COOH O-OH Fe2.

~'~

°, 1

CHs'CH2"CH'CH"CH'CH:CH'CH2-CH-'CH"(CHa)v'COOH

0-0" L"

(L;:o.j)

(LO0')

(LOOH)

I FeS.

(H02) 1

Co~Juslmt|on

CHs'CH2".CH'CfltCH'CH=CH'CH2-CH=CH"(CHa)T "COOfl

LH

oxidation is induced by radicals. It was found that the primary radical OH • of water radiolysis, certain organic radicals, but also metal ions, such as Fe 2+, function as initiators or promotors of a radical chain mechanism leading to lipid peroxidation. Lipid peroxidation is of great importance in different areas. It is of biological and of medical relevance, since it forms the basis of a number of diseases and has been discussed in the context of carcinogenesis and human aging (see, for example, Ref. 10 and Section VI of the present article). It is also of great nutritional consequence. Autoxidation of lipid-containing food not only produces offensive odors and flavors but can also decrease the nutritional quality and safety by the formation of secondary products in foods after cooking and processing [18]. There have been many efforts to clarify the mechanisms of lipid peroxidation in view of its great practical importance. The results have been summarized in numerous reviews (e.g., Refs. 16-21). The present article concentrates on the main aspects of the radiation induced process. Lipid peroxidation is most efficient with lipids possessing polyunsaturated fatty acid residues. Figs. 2A and 2B show a simplified (and partly hypothetical) reaction scheme of the peroxidation of linolenic acid with three isolated double bonds. Peroxidation is initiated by O H , radicals. The fatty acid LH by way of hydrogen abstraction is converted into the radical L .. Though there is evidence that O H radicals hardly discriminate between the different Catoms of the alkyl chain, the unpaired electron is found in the double allylic position as shown in Fig. 2A. This is believed to occur by intramolecular and intermolecular (from neighbouring molecules inside the membrane) hydrogen abstraction [22]. In a similar way the formation of the diene Lco,j. may be understood, which

) (02¢H20)

OH- ¢ CHs-CH2-CH-CH:CH-CH:CH-CH2-CH:CH-(CH2)T-COOH (LO')

o

CHs-CH~ ¢

I

LH 1 >L"

[

HO,',C'CH:CH'CH'-CH-CH2-CH-'CH"(CH2)v-cOOH

) initiation of a further radical chain CHs'CH2"CH'CH:CH'CH-'CH'CH2"CH-"CH" (CHa)~,-COOH (LOOH) according to ['~ O-OH CHfCH~ Fig. 2. Reactionschemefor OH--inducedperoxidationof linolenicacid and the formation of ethane (modified after Ref. 19). (A) Initiation of the radical chain mechanismby hydroxylradicals and the formation of lipid hydroperoxides.(B) Elongation of the radical chain by Fe2+ and/or HO2" radicalsand the formationof ethane.

107 shows strong absorption at about 280 nm. Patterson and Hasegawa [22] observed the time dependence of this process by pulse radiolysis of aqueous sodium linoleate. The same system was used to measure the addition of oxygen, i.e., the formation of the peroxyl radical LOO °, by utilizing the absorption of this species below 260 nm [23]. The rate of the partial reactions considered so far approaches that of diffusion controlled reactions (see section II-E). The reactivity of the peroxyl radicals LOO • towards suitable hydrogen donors, such as oxidizable polyunsaturated substrates, is smaller. Nevertheless, rate constants of the order of 100 M-1 s-1 were observed for this process [20]. In this way stable lipid hydroperoxides are formed at the expense of further lipid radicals L °. The latter may initiate a chain mechanism by repetition of steps 2 and 3 of the reaction scheme. Thus, the peroxidation process will continue until the species L o are eliminated by radical-radical interactions (see below). The kinetic chain length, v, of radiation induced peroxidation may be defined by

u=

n u m b e r of 0 2 molecules consumed initiating O H radical

converting 0 2 ° / H O 2 • radicals to 0 2 and H 2 0 2 . There is, however, an alternative way O f . , / H O 2 • radicals might augment lipid peroxidation. They could indirectly contribute to the initiation of the process. This might happen via O H - radicals produced by a metal-catalyzed Haber-Weiss reaction [17,19]: 0 2-

+Fe

3+ ~

0 2 +Fe

2+

Fe 2+ + H202 + H + -~ O H • + Fe 3+ + H 2 0

(12) (13)

Eqn. 13 is the classical Fenton reaction. There seems to be general agreement that HO 2 ° radicals are not able to initiate lipid peroxidation in a direct way. The relative contributions of 0 2 • , / H O E • radicals to the initiation (in the presence of transition metals) and to the promotion of lipid peroxidation (by decomposition of hydroperoxides) remains still to be clarified. The kinetic length, o, of the radical chain is limited by bimolecular reactions of the different radical species. It is usually assumed that various types of radicals (i.e., L . , L O 0 °, etc.) may react with each other according to

(11)

El° + L k-

Thus, v is equivalent to the number of cycles of the reaction scheme shown in Fig. 2A. v is enlarged in the presence of Fe 2÷ and of other transition metal ions. It was found that lipid hydroperoxides are decomposed by metal catalysts leading to the gaseous end products ethane or pentane [19,24]. Alkoxy radicals LO ° are assumed as intermediate products which break down to fatty acid aldehydes and to alkyl radicals (c.f. Fig. 2B). The latter may initiate a new radical chain according to Fig. 2A. Therefore, Fe 2÷ may be designated as a promoter of lipid peroxidation. In the presence of hydrogen peroxide, Fe 2+ - via the generation of OH • radicals - may also initiate this process (c.f. Eqn. 13). We have found that the augmentation of radiation induced lipid peroxidation by Fe 2÷ may also be observed at the level of membrane transport (C. Barth and G. Stark, unpublished data). A similar role as for Fe 2÷ has been suggested for HO 2 ° radicals [25,26]. Though different authors have argued against the ability of HO2" to react with hydroperoxides [27,28], such a reaction would explain the strong pH-dependence observed at the influence of lipid peroxidation on carrier mediated K+-transport across lipid membranes (c.f. subsection IV-B). The promotion of lipid peroxidation by HO 2 ° according to Fig. 2B was previously reported by Petkau [29,30], who found a reduced yield of peroxyl radicals LOO • in the presence of the enzyme superoxide dismutase. This enzyme has been suggested to reduce oxidative cellular damage by

kik

..

,

r n o n r a a x a l product,

(14)

where L i • and L k ° represent identical (i = k) or different (i #= k) radical species. Figs. 2A and 2B only show the main features of one important pathway of lipid peroxidation. Another important reaction sequence leads - via lipid endoperoxyl radicals - to the end product malondialdehyde (see e.g., Ref. 19). One may ask for the general consequences of lipid peroxidation for membrane transport. Two different modes of action are immediately apparent and have been confirmed by studies on lipid model systems (c.f. Sections III and IV): The accumulation of polar products (lipid hydroperoxides, aldehydes, etc.) gives rise to an increase of the dielectric constant of the membrane interior. This has a strong bearing on the movement of charged particles across the membrane. The second consequence results from the bimolecular reactions terminating the reaction chain (Eqn. 14). Though the chemical nature of the nonradical end products have not been analyzed in detail, such reactions may be expected to lead to cross-linking of lipid molecules. This will influence the microviscosity of the membrane interior and will finally modify its diffusion properties. H-E. The inverse dose rate effect

Lipid peroxidation - as outlined in Section I - shows the strange phenomenon of an inverse dose rate effect, i.e., the radiation effect, at constant absorbed dose,

108 increases with decreasing dose rate. Inverse dose rate effects were found to be a direct consequence of certain types of radical chain mechanisms and have been detected many years ago throughout kinetic studies in the chemistry of rubber and related materials [31,32]. In the case of radiation induced peroxidation of fatty acids and lipids, the phenomenon was first described by Mead [33] and was subsequently studied by many different authors [29,30,34-44]. In view of the potential importance of lipid peroxidation for the evaluation of the genetic and of the carcinogenic risk following radiation exposure, the mechanistic aspects of the inverse dose rate effect will be treated in some detail. The phenomenon is usually explained on the basis of a simplified reaction scheme derived from Fig. 2A. The radical chain may be subdivided into three different steps, namely initiation, propagation and termination. The three steps are defined as follows: Initiation (by a radical species X . , e.g., O H . ): LH+ X.--~L. +XH,

(15)

Propagation of the chain: L. + 0 2~ LOO. LOO. + LH

kp

~LOOH+ L.,

(16) (17)

kt

-dO2/dt =

R i + kp L H

(18)

Eqn. 18 represents a special case of Eqns. 14, valid for sufficiently high oxygen concentration so that the concentration of L , may be neglected against that of LOO ,. In order to prove the existence of an inverse dose rate effect at radiation induced lipid peroxidation, one has to show that the kinetic chain length, v, defined via Eqn.11 is reduced at high dose rates when compared to low dose rates (at constant total absorbed dose). This may be visualized on the basis of Eqns. 15-18 as follows: Neglecting the terminating reaction (18), a single initiating event, because of the unlimited propagation, would peroxidize the total lipid available. In reality the kinetic chain length, v, is limited by the bimolecular termination reaction. The rate of this reaction proceeds with the second power of the concentration of cadicals LOO -. Assuming a constant total number of initiating events (i.e., a constant absorbed dose), the importance of the termination Eqn. 18 - because of the second power dependence of its rate - increases with the rate of the events applied. Shorter chain lengths

(Ri)l/2//(2kt) 1/2,

(19)

Combining Eqns. 11 and 19, one obtains v=

[dO2/dtl kp LH R--------~ =1+ (2kt)l/----~ (Ri)l/2

(20)

For kpLH >> (2ktRi) I/2, the kinetic chain length, v, is inversely proportional to the square root of the rate of initiation, whereas for kpLH 100). To measure D37(Ar). the cuvette used for bilayer formation was flushed with argon to reduce the oxygen concentration to the order of 1%; (c) The /)37 dose was found to increase by about one order of magnitude in the presence of the radical scavengers t-butanol, formate, or hydrogen peroxide; (d) The sensitivity is also reduced, if the membrane is formed from lipids containing unsaturated ~atty acid residues (e.g., dioleoylphosphatidylcholine instead of diphytanoylphosphatidylcholine, c.f. Fig. 15). The experiments may be explained by assuming a combined action of OH • and HO2 ° radicals to be responsible for the channel inactivation. The inhibitory action of the radical scavengers t-butanol and formate as well as the protection of the channels by double bonds of the fatty acid residues of the lipids indicate the importance of OH ° radicals (c.f. sections ll-C and II-D). The participation of HO 2 ° radicals at the channel inactivation may be concluded from the absolute requirement of 0 2 in combination with the pronounced pH-dependence and from the inhibitory action of H20 2. In the presence of the latter, the primary radicals ea~ and H ° are converted into OH ° radicals at the expense of oxygen radicals formed according to reactions (2) and (3). The reduced sensitivity of the channels observed under the condition of an increased OH • and a reduced HO2 ° concentration (in combination with the requirement of OH • , see above) shows that the simultaneous presence of both types of radical is required for channel inactivation. In order to identify the chemical groups responsible for the inactivation of the channel, experiments with different chemical analogues of gramicidin A were performed, some of which are shown in Fig. 12. Among the essential amino acids, those possessing aromatic side chains or sulphydryl groups are usually considered to be most reactive towards an attack by water radicals. This was also confirmed in the case of gramicidin A. The targets are represented by the four tryptophan residues of gramicidin A. The residues, according to Urry's model (c.f. Fig. 13), are situated near the channel mouth, where they are exposed to radical attack from the aqueous phase. The importance of the tryptophan residues is shown by experiments with analogues of gramicidin A, which have ~he tryptophan residues replaced either by phenylalanines (gramicidin M), by naphthylalanines (compound GN, c.f. Fig. 12) or by tyrosines (compound GT). A 50-fold reduction of the

118 sensitivity was found for GT and a more than 1000-fold reduction in the case of the two other compounds [79,81]. To study the influence of the single tryptophan residues at the four different positions, experiments with analogues were performed, having one tryptophan residue in position 13 (GNg']m5), or two tryptophan residues in positions 11 and 13 (GN 9'1s) or in positions 9 and 15 (GNna3), respectively (c.f. Fig. 12). The same radiation sensitivity, expressed by the value of the D37dose, was observed for the compounds with two tryptophan residues as compared with normal gramicidin A. In the case of GN 9'1u5 the sensitivity was lower by a factor of 8. There is, however, one important difference between the analogues. In the case of normal gramicidin A inactivation is complete, i.e., the conductance, at sufficiently high radiation doses, corresponds to the conductance of a pure (unmodified) lipid membrane. For the analogues with one or two tryptophan residues, a small but significant (virtually) radiation-insensitive final conductance was observed [81]. The same behaviour was found on the level of the single ion channels. The analysis of the single channel fluctuations in the presence of normal gramicidin A resulted in the same distribution of conductance amplitudes and the same open times of the channels before and after irradiation. This was interpreted as an 'all-ornothing-behaviour'. Damaged channels are 'electrically silent', i.e., only intact gramicidin channels contribute to the membrane conductance observed [79]. On the other hand, a broad distribution of conductance states of lower amplitude was found for the analogues with one or two tryptophan residues per monomer, (c.f. Fig. 16), i.e., the conductance of damaged channels is different

iated

0.15

from zero in these cases. The mean conductance of irradiated GNg"11'15-channels is changed by 17%, that of GNga5-channels by 43% [81].

Kinetic analysis of channel inactivation The observation of identical conductance states before and after irradiation of normal gramicidin A can be explained most easily by assuming that a radical attack at a single tryptophan residue is sufficient to convert the channel into a state of virtually zero conductance. Intermediate conductance states should be present, if more than one tryptophan residues were to be modified by radicals. The conductance of the intermediate states should correspond to those observed for GN 9'11A5 and for GN9'lS-channels. The finite conductance values found for the latter indicate, however, that the presence of at least two further tryptophan residues (not directly attacked by radicals) is essential to obtain the state of virtual zero conductance. The presence of these residues, which are in close sequential and spatial proximity, might enable an efficient energy transfer between the tryptophan residues of a monomer, i.e., a radical-induced change of one residue could give rise to a chemical modification of the other tryptophan residues. The hypothesis was tested by a quantitative analysis of the inactivation process following continuous radiolysis or pulse radiolysis. The results of both types of experiments were compared with a simple model of inactivation, which is based on the following assumptions [79,81]: (1) Inactivation is started by a sequential reaction of a radical R1 (presumed to be OH, or a secondary radical produced by OH, such as CI~-) and a radical R2 (presumed to be HO2) at a single tryptophan residue of gramicidin A:

)1 •~

A + R1 --* A'

(35)

A' + R2 --* B

(36)

o.1 O.O5

(2) The species B initiates a series of radical transformations 20

40 60 80 .A./pS Fig. 16. Probability P(A) of the observation of a conductance fluctuation A in the presence of single channels of the gramicidin analogue GN 9"15 before and after exposure to X-rays. The membranes were formed from monoolein/n-decane in the presence of 1 M CsCI. A nominally 10 -6 M solution of GN 9a5 in 1 M CsCI (pH 3) was irradiated with 2000 Gy, a radiation dose sufficient to damage virtually all GN 9a5 molecules. A small amount of tiffs solution was added to the aqueous solutions surrounding the membrane and the resulting fluctuations were observed as a function of time at a constant voltage of 50 mV. The total number of analyzed channels was 1180 (nonirradiated) and 1278 (irradiated). The mean value, A, of conductance fluctuations was 44.2 pS (control) and 25.2 pS (irradiated). (From

Ref. 81.)

B..-~... --,.R,

(37)

which, via an unknown number of intermediate products, finally leads to the radiolyzed state R of gramicidin. The latter depends on the number of tryptophan residues. (3) The species A represent nonconducting precursors of the channel. Activation of the channel proceeds by association of two precursor molecules: K A+A~AA

(38)

119 The same reaction is assumed for A' and for R. The species AA, AA' and A'A' represent the conducting (open) state of the channel with the same unit conductance, while the species AB, A ' R and RR (for normal gramicidin A) are treated as nonconducting. Eqn. 38 depending on the model of channel activation - is equivalent to Eqn.34 or describes the first step of the aggregation phenomenon shown in Fig. 14. (4) The lifetime of the radiation induced radicals R1 and R2 is determined by scavengers S1 and $2. For the radical R2 a mutual deactivation (Eqn. 41 was taken into account in addition to the scavenging process: R1 + S1 --, products

(39)

R2 + $2 ---,products

(40)

R2 + R2 ---,products

(41)

Eqns. 40 and 41 were suggested by Rabani and Nielsen [102] and by Bielski and Allen [103], who found that the time dependence of HO 2 radicals is adequately described by a combination of both equations. $1 and $2 represent impurities in the aqueous phase, as well as reaction partners at the membrane/water interface. In spite of the underlying simplifying assumptions, the model was found to provide a satisfactory description of the shape of inactivation curves. This includes the shoulder curves (and their modification by radical scavengers) found at continuous irradiation of the membrane at constant dose rate (c.f. Fig. 15) as well as the nonexponential time dependence of the channel inactivation following a single pulse of 14 MeV electrons (c.f. Fig. 17).

o

.5

v

17

I

I

O0OClUUO0 |

2

time/s Fig. 17. Time dependence of the inactivation of gramicidin channels in diphytanoyllec?.thinmembranes followinga singlepulse of 14 MeV electrons at two different radiation doses (0.84 Gy and 1.54 Gy). The higher the radiation dose, the shorter is the time range needed for the inactivation. At 21.7 Gy the inactivation is complete within less than 50 ms (data not shown). The solid lines were calculated according to the model of channel inactivationdiscussed in the text. (From Ref. 79, with permissionof Taylor & Francis, Ltd.)

Consequences of irradiation for the channel structure The study of the inactivation of gramicidin channels may be used to test the current views on the channel structure. The membrane conductance, h, is determined by the product, A N o, of the single channel conductance, A ,a~d of the number, N o, of open channels. The foregoing discussion has shown that the value of A, depending on the number of the tryptophan residues per monomer, is reduced in the radiolyzed state of gramicidin. The main effect of ionizing radiation, however, applies to the number of open channels. This is concluded from a comparison of the radiation effect on the membrane conductance, A and on the single channel conductance, A. For the analogues G N 9ALAS, G N 11A3 and G N 9A5 the effect on No was found to be considerably larger than the effect on A. N O is determined by the rates of channel formation and dissociation. The dissociation rate, estimated via the mean channel life time, is hardly influenced by irradiation. Therefore, a strong reduction of the formation rate of open gramicidin channels is believed to represent the main part of the radiation effect. This holds irrespective of the channel structure and of the nature of the opening and dosing events. The radiation effect has been shown to be due to the presence of the tryptophan residues of gramicidin A, which are presumed to be chemically modified. Therefore, any molecular model of the channel structure has to explain, how, by modification of the tryptophan residues, a reduction of the formation rate can be achieved. The phenomenon described appears difficult to be explained in the frame of the monomer-dimer hypothesis (Eqn. 34). The radical-induced modification of the Trp-residues occurs at the C-terminal end, near the membrane/water interface. For the association of monomers to dimers, however, the N-terminal end located in the membrane interior is responsible. The aggregational model of channel opening, on the other hand, is based on dimer-dimer interactions for which Trp-Trp contacts have been suggested to be of great relevance [100,1011. Thus, the radiation induced reduction of the rate of channel formation (i.e., the decrease of the aggregation rate) becomes plausible. The second property influenced by ionizing radiation is the single channel conductance. So far, no defilfite structural interpretation of the effect is available. The radiation chemistry of tryptophan has been studied repeatedly including reactions of the amino acid with primary water radicals and an analysis of the products of radiolysis [104-108]. The initiation of the channel inactivation, given by Eqns. 35, 36, agrees with an early study of the effect of X-rays on tryptophan in aqueous solution [105]. Intramolecular radical transformations (e.g., charge transfer) in tryptophan containing peptides, as postulated by assumption 2 of the inactivation model,

120 has been found to occur over considerable distances [109]. A simple ad hoc explanation of the reduced single channel conductance is the assumption of positive charges (or of dipole moments of suitable orientation) at the channel mouth introduced by the reaction products of tryptophan radiolysis. This could represent an electrostatic barrier for the entrance of cations into the channel. A definite answer, however, requires a chemical characterization of the products of gramicidin radiolysis, which has not been performed so far. The study on the gramicidin channel indicates that the important role of the amino acid tryptophan reported for the inactivation of water-soluble enzymes [110-112] is also valid for membrane proteins.

V. Biological implications The studies dealt with in the preceeding paragraphs were performed with well-defined model systems. Therefore, a direct transfer of individual results to biological systems of considerably larger complexity, is not allowed. Nevertheless, the general principles should be also valid for cellular membranes. The important points may be summarized as follows: (1) primary water radicals as well as certain secondary radicals represent an important source of deleterious effects not only for cytoplasmic proteins but also for the components of biological membranes. Some types of radicals are able to penetrate into the membrane interior and may react either with the lipid matrix or with integral membrane proteins; (2) the radicals may influence the function of membrane proteins either indirectly - via a modification of the surrounding lipid matrix - or by a direct interaction with the protein in question. Lipid peroxidation - by a change of the dielectric properties of the membrane interior - is of great consequence for the rate of movement of charged particles across the membrane interior. Though the latter was shown for carrier-ion complexes only, the same behaviour may be expected for all kinds of ionic movements including the movement of charged side groups of proteins; (3) ion channels may represent radiation sensitive structures, the importance of which have been underestimated so far. A wealth of different cellular phenomena depends on the proper functioning of the different ionic pathways through biological membranes. Therefore, the study of the model channel formed by gramicidin A should be extended to channels of native biological membranes. Thereby the patch clamp technique may be applied to study radiation effects on the level of single channels; (4) there is unequivocal evidence for an inverse dose rate effect following irradiation of lipid model systems under a variety of different conditions. The increase of the radiation damage with decreasing dose rate has been

observed over many orders of magnitude. A significant radiation effect was reported at radiation doses below 1 rad = 0.01 Gy at sufficiently small dose rates [29,30]. This means that the effect of lipid peroxidation is detectable even at the natural level of ionizing radiation. Inverse dose rate effects have also been observed at the cellular level in the form of radiation induced neoplastic transformations [113,114] or mutations [115]. In one case an increased rate of leukemia induction in mice was reported after protracted administration of 224Ra [116]. There is no direct evidence for a relation between the different biological effects and the lipid effect. A (so far) hypothetic correlation may, however, be obtained, if mutations are considered as the basis of neoplastic transformations and of cancer. There seems to be increasing evidence about a mutagenic effect of certain products of lipid peroxidation (for a review c.f. Ref. 117). A possible pathway for the interaction between a lipid radical LX- and DNA is given by Eqn. 42: [ DNA-adducts LX. + DNA -, LXH + DNA. --, ~ DNA-strand-breaks [ DNA-protein-crosslinking

(42)

Similar kinds of DNA damage are possibly induced by certain final products of lipid peroxidation such as malondialdehyde. By way of Eqn. 42, an inverse dose rate effect, originating from the lipid component of biological membranes, could be transferred to the genetic substance. The experimental evidence for the outlined hypothesis is scarce. In view of the utmost practical importance the hypothesis would have for the radiation protection (i.e., for the evaluation of the genetic risk and of the cancer risk at small radiation dose), the study of the inverse dose rate effect deserves, however, any attention. Current estimates of the cancer risk are largely based on the experience of the survivors of the atomic bombings of Hiroshima and Nagasaki, i.e., from a radiation exposure of very high dose rate. Extrapolation to lower dose rates would substantially underestimate the radiation risk, if cancer induction shows an inverse dose rate behaviour. Acknowledgments The author would like to thank his collaborators M. Str~issle and C. Barth who contributed an important part of the experiments reviewed. The author is obliged to Dr. A. Henglein and to Dr. W. Schnabel for giving us the opportunity to perform pulse radiolysis experiments at the Hahn-Meitner-Institut (Berlin). The experiments at this institution would not have been possible without the continuous help by M. Wilhelm. The study was

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