Indian Journal of Experime ntal Biology Vol. 39. April 200 1. pp. 29 1-309

Review Article

Radiation induced peroxidative damage: Mechanism and significance *

Anjali Agrawal & R K Kale** School o f Life Sciences. Jawaharlal Nehru Uni versity. New De lhi 110067. India

An interes t has been generated in free radica ls aftcr the di scovery o f superoxide di smutase. These free radicals cause a number o f di seases and arc in vo lved in the detrimental effec t o f io nizing radiatio n. Efforts have been made to understand the ir role in damage and death of the (;e ll using lipid peroxidation process. Lipid peroxidatio n is an important e ffect o f radiation on membranes. which apart from DN A. are critical targets o f radiati on act ion. Thi s paper addresses the basic mechanism o f radi atio n induced lipid perox idJ tio n. Various fac tors. which dete rmine the mode and mJg nitude o f li pid peroxidati on. are also di scussed . Lipid perox idation is shown to have impo rtance in understanding the modificati ons o f radiation e ffec ts. Efforts are made to sho w similar ities between radiolytic and non-radiolytic lipid perox idation. Recent find ings re lated to the close link between radi atio n-induced lipid peroxidation and apo ptosis are likely to open new avenues for future research and to develop new approaches for radio modiflcation of bio logical effects.

Since living beings are built up of molecules, all biological reactions have to be molecular, making life a molecular phenomenon I. While understanding this phenomenon , an overemphasi s was given on the chemical reacti ons which were known to involve polar reactants and intermediates, generated from the heterolytic dissociation and formation of chemical bond . Free radicals formed by homolytic dissociation of chemical bonds are ignored bybiologists and clinicians. Thus, biology is dominated by the chemistry, based on ionic theory of dissociation . However, there is an explosion of interest in free radicals after the discovery of superoxide dismutase (SOD), an enzyme specific for the catalyt ic removal of superoxide (0;- ) free radicals. Free radical generation in living systems occurs mainly through three-electron reduction of molecular oxygen, instead of a fou r-electron reduction. Several redox processes (e.g., Fenton reaction) and enzymatic reactions (e.g., xanthine oxidase) contribute to free radical production . Apart from biochemjcal sources, free radicals are also generated due to exposure to radiation. Detrimental effects of ionizing radiation on living systems are mediated through these free radicals. Free radicals interact with cellular

**Correspondent author. Fax: 91-1 J -61 87338 *Thi s paper is dedicated to Dr A. Ramesha Rao. Professor o f Cancer Biology. School of Life Sciences. Jawaharl al Nehru Uni versity. New De lhi 110 067. who made an outstanding contribution to radiation and cancer biology. His work on chemoprevention has placed Ind ian plants and phytochemieals on the world map o f cancer research.

components and bring about a change in structure and function of biological molecules, which cou ld lead to damage and death of cell. ow it is well established that free radica ls also cause a number of diseases . Efforts have been made to understand the generation of free radicals, damage induced by them and the mechanism of damage. The lipid peroxidation process is extensively used for this purpose, mainly in non­radiolytic processes. Though known to be a free­radical chain reaction, lipid peroxidation is not well exploited to understand the biological effects of radiation.

Apart from DNA, membranes are also considered to be critical targets of ionizing radiation effect. Bacq and Alexander2 had proposed that a significant contribution to radiobiological effect of ionizing radiation is due to cell membrane damage. Several studies have supported the idea that membrane damage induced by radiation is the critical event3

-8

.

Kerr el 01.9 have suggested that damage to membrane organization is an initial step in triggering cell death. A correlation has been observed between unrepaired membrane damage and loss of colony formi ng ability in cells 10- 12, the breakdown of nuclear membrane and chromosomal condensation and damage to the organization of mitotic spindles 13

, interphase death in non-proliferating ce lls and the di sorgan izati on of mcmbrane system l4

. Since lipid peroxidation is one of Ihe important effects on biological membranes 15, the studies in lipid peroxidation can provide important information about detrimental effects of radiation. In addi tion, the process can be utilized to understand the

292 INDIAN J EXP SIOL. APRIL 200 1

pathways involved in radiomodification by chemical agents. Therefore, an attempt has been made to discuss some important mechanistic aspects and the usefulness of radiation-induced lipid peroxidation. Efforts are also made to compare the radiolytic and non-radiolytic lipid perox idation.

Cellular dall/age and lipid peroxidation-Lipid peroxidation is a hi ghly destructi ve process and alters the structure and functi on of cellular membrane l6

. It is involved in a number of diseases and in poisoning of several toxins l7

. Disrupted tissues are known to undergo lipid peroxidation at a faster rate than normal ones. Lipid peroxidation, therefore, can be used as a measure of ox idative damage. Perox idation brings about change in structure, fluidity and permeability of membranes I8.21

; inactivates a number of membrane­bound enzymes and protei n receptors22

-26

; induces swelling and alterations of respiratory functions27

;

causes loss of -SH groups from the membrane-bound proteinsI5.28

; mediates DNA damage29; and alters

R A transport from nucleus to cytoplasm3o.

Carcinogenesis is also related to lipid peroxida­tion 31

-35. Radiation-induced apoptosis and lipid

peroxidation are closely linked36.

Lipid peroxidation: a chaill. reaction-Spontaneous ox idation of lipid molecules in membranes by oxygen at room temperature is termed as lipid perox idation . It is a free radical chain reaction and involves three distinct steps, i.e., II1ltl atlOn, propagation and termination as shown in the following scheme:

Initiation

H20 ~ HO' , H' , e~q' 0;- ,H 2 , H 20 2

LH+'OH~L'+H20

Propagation

L'+02~LOO'

LOO'+LH~LOOH+L'

Termination

L'+L'~L-L

LOO' +LOO' ~LOOL+O:!

LOO'+L'~LOOL

( I )

(2)

(3) (4)

(5 ) (6) (7)

Removal of a hydrogen atom homolytically from a methylene carboll of an unsaturated fally acid (LH) can initi ate lipid peroxidation. The ground state or

polyunsaturated fatty acids (PUFA.) is of singlet multiplicity and their reacti ons with oxygen are forbidden since the ground state of oxygen is of triplet multipliciti7

. The lipid peroxidation reaction thus involves a mechanism that circumvents the spm ban-ier. The possibility to overcome the spin restriction is via free radicals.

Since biological systems consist of about 60-80% water, to a great extent, the biological effects of ionizing radiation are mediated through radiolysis of water (reaction I). A cell contains about 1013

molecules of water. A dose of 1 Gy (=0.6 X 1018 eV Kg- I) may cause 2 x 105 ionizations Kg- I. Therefo re, 2 x 105 ionizations are expected to be formed in a cell mass of IO-9g. These ionic products (ion pairs) are very active and get converted into free radicals. Among these, 'OH radical is considered to be responsible for initiation of radi at ion-induced lipid peroxidation38

-4o

. "OH radical is hi ghly reactive and has sufficient energy to abstract an allyli c (methylene) hydrogen from LH. Therefore, 'OH radical initi ates radiolytic and also non-radiolytic lipid peroxidation. In an artificial lecithin bilayer, the rate constant for removal of hydrogen is found to be about 5x 10 M-I S-I (ref. 4 1).

Involvement of 0;- in lipid peroxidation is also

suggested42. It is important to note that 0;- can be

generated within cell s through endogenous metabolic activities and by intracellular redox cycling of exogenous compounds. There is, however, still an uncertainty about its role in in itiation of lipid peroxidation . Radiolytic studies have consistently

shown that 0;- does not initiate lipid peroxidation in

pure PUFA38.43

.44 . 0;- is insufficiently reactive to

abstract the hydrogen from lipids. Therefore, direct

involvement of 0;- in initiation of lipid peroxidation

can be ruled out. Although the protonated form

(HO;) (pKa=4.8) is a stronger ox idant than 0;- and

can initiate lipid peroxidation4.J, at physiological pH

0;- remains almost entirely in the unprotonated form

(reaction 8):

... (8)

However, HO; IS ava il able In lysosomes,

phagosomes and adjacent to an ioni c surfaces for initiation of li pid peroxidation (reac tion 9). \3u t no

AGRAWAL & KALE: RADIATIO I DUCED PEROXIDATIVE DAMAGE: MECHANISM & SIG IFICANCE 293

information is available regarding the role of HO; 111

such a microenvironmental condition:

. . . (9)

Remember that

peroxidation V/Q

0 ·-2

the

indirectly influences lipid

Fenton/Haber-Weiss re-action 40-42.45:

0 ·- + Fe3+ --7 Fe2 + 0 2 2 (10)

20;- + 2 H+ ~ H20 2 + 0 2 ( II )

Fe2+ +H202~Fe3+ +OH-+HO· ( 12) Fe2+ +LOOH~FeJ+ +HO-+LO· ( 13)

Generation of 0;- by any source, in the presence

of transition metal ions, particularly iron ions, can lead to formation of ·OH radical and in turn may

initiate lipid peroxidation. Thus 0;- certainly plays

an important role in initiation of lipid peroxidation through the redox cycle of transition metal ions.

Fenton chemist ry is not the on ly possible pathway

for transformation of 0;- into more reactive species.

The reaction with hypochlorous acid (HOC!) is also suggested as a source of HO· radicals. In addition, the

reaction of 0;- with nitric oxide (NO) produces

peroxynitrite which can lead to oxidation and nitration of biological molecules and upon single electron reduction yields nitrogen dioxide46. Hypochlorous

acid reacts with 0;- to generate HO· radica ls47 .

(reaction 14):

.. . (14)

The reaction 14 is an analogue of Haber-Weiss reaction, but it does not require metal ions as catalysts. It proceeds spontaneously with a high rate constant (k= 7_5 x 106 dm3m-'s-')48 and is termed as Bielski reaction46.

The reaction of 0 ;- with NO generates

peroxynitrite (ONOO-; k= 6.7 x 109 dm3mor's-')49 (reaction 15) which mediates cell injury and various inflammatory disorders50

.5' as a result of oxidation and nitration of biological molecules52. Peroxyni trite is stable in basic solution, but upon protonation isomerizes to nitrate (reaction 16). In addition, nitrogen dioxide (N02) or ·OH (reac tions 17, 18)

formed from the reaction of peroxynitrite is suggested to be involved in detrimental effect of radiation53. Nonionizing radiation (UYB) has been shown to act as a potent stimulator of nitric oxide synthetase (NOS) and xanthine oxidase (XO) in human endothelial cells, leading to release of NO and ONOO- and in turn the cytotoxic effects54. However the reaction 18 is thermodynamically favoured over reaction 17:

0;- +NO~ONOO- (15)

ONOO-+H+~ONOOH~ NO ) +H+ ( 16)

ONOO-+e-+W~OH"+ NO ) (17)

ONOO-+e-+H+~ NO; +OH- (18)

Thus, the reaction with hypochlorous acid and nitroxide can be regarded as an alternative pathway

for conversion of 0;- into more reactive species.

Lipid peroxidation can be initiated by various

agents that do not involve 0 ;- . Importance of iron­

centered radical in lipid peroxidation was proposed around 35 years ag055. Since then, extensive work has been carried out using ascorbic acid, NADPH and Fe2+-dependent lipid peroxidation to understand the redox cycling of iron. By definition, iron ions are themselves free radicals and react with oxygen to give perferryl (reaction 19)/ferryl (reaction 20) ions called ferrous dioxygen complexes:

Fe2+ +02HFe2+-02HFe3+ + 0;­

Fe2+ +02H Fe02+

(19)

(20)

These ferrous dioxygen complexes can initiate lipid peroxidation55

.56. Interestingly, the rate of lipid peroxidation has been found to be maximal when the ratio of Fe2+ to Fe3+ is 1: 1 (Ref. 55). The enhanced rate is attributed to Fe2+-oxygen-Fe3+ complex as an initiating species. Iron also increases radiation­induced lipid peroxidation. Moreover, ferrous has been found to be released from proteins on irradiation37. Therefore, it is quite possible that iron complexes of oxygen may also be contributing to lipid peroxidation through secondary initiation in radiolytic systems and result in new reaction chains of lipid peroxidation. Now it is quite clear that any free radical which has sufficient energy can abstract a hydrogen atom from the methylene carbon of unsaturated fatty acids and initiate lipid peroxidation.

294 INDIAN J EXP l3I Ol , APRil 2001

Abstraction of hydrogcn from methylic (-CHr) group results in formation of a carbon-centered free radi cal (-·CH-) which reacts rapidly with molecular oxygen (k = I OR_ I 0 10 M'I S'I) to generate peroxyl

radical ( LO; ; reaction 3)57, Peroxyl lipid (LO;) is

reacti ve and is able to abstract allylie hydrogen (reaction 4) from the side chain of adjacent PUFA to yield a lipid hydroperoxide (LOOH) and lipid radi cal (L·) (k=31 M'I S' I; Ref. 58) . ewly formed lipid radicals as shown in reaction 3 interact with O2 and close the self-propagating cycle. As long as lipid molecul es and oxygen are ava il able, the propagation is likely to continue. Lipid perox idation process necessarily requires molecular oxygen for its propagation5

!). LOOH is the predominant product of the propagation cycle. Even though chain propagation is relatively slow, the concentration of lipid within the membrane can make propagati on significant41

• It is important that the rate of initiation of lipid peroxidation is quite fas t, but the propagation (formation of hydroperoxides) is considerably slow.

Lipid peroxidation has no intrinsic metal ion requirement. Nonetheless, iron compounds can increase the rate of propagation of lipid peroxidation dramatically by decompos ing lipid hydroperoxides

(LOOH) to reactive alkoxyl (LO·) or peroxy l (LO; )

radicals (reactions 21, 22), which can initiate new reaction chains6o

.6 1

:

LOOH+Fe2+ ---1LO·+OH'+Fe3+ LOOH+Fe3+ ---1 LOO·+H+ +Fe2+

(2 1 ) (22)

However, at higher concentration , iron ions shi ft from being repeatedly cycled as catalysts to serve as stoichiometric reactions and can ex hi bit dominant chain termination antiox idant behav ior attributed to following reacti ons62

. The antiox idant behaviour of iron is not seen at low concentrations63

.6.J:

Fe2+ + LOO· + W ---1 Fe3+ + LOOH (23) Fe2++·OH+H+---1 Fe3++H20 (24)

Fe2++LO·+W---1 Fe3++LOH (25)

(26)

These reactions are very fa. t and their imparl ance has now been emphasized . a t much is known about the dual effect of iron in radiation-induced lipid perox idation.

The free radical chain reaction propagates until two free radical s destroy each other to terminate the chain. In the termination phase, two free radicals combine to yield a non-radical product. As shown in the reac ti ons 5-7, termination can occur due to self-reaction of two L· radicals (k= 108 M'I S' I) or two LOO· radi cals (k-107 M' I S' I) and cross terminati on of LOO· radicals with L· radical (k= 107M' I S' I; Ref. 41 ).

During the early phase when concentration of LOOH is low, the generation of L· occurs primarily via reaction 2, because the rate of propagation process (reaction 4) is slow compared to initi ation (reaction 2). As time lapses and LOOH accumulates, the generati on of LO is increased during the self­accelerating propagation reacti on (reactions 3, 4) . Therefore, formation of L· via reac ti on 2 loses its importance for initi ation of lipid perox idation over a time in favor of L· formed by the self-accelerati ng reactions which steadily increase in rate with time.Jl. Since ·OH is continuously formed during irradiation, L· may not lose its importance in un initiation phase of lipid pcroxidution in radiolytic systems.

In free radical chain processes there are many potenti ally competing reactions for 1I1ltIatlon, propagation and termination . Both the relative as well as the absolute concentration of all reactions play an important role. However, a balance between the competing reactions depends strongly upon absolute

. 6? R d' I . concentratIons -. a 10 ytlc systems are more complex, as reactants are consumed, inacti vated or decomposed and many products are formed simultaneously. Being a free radical phenomcnon, th is could be true for radiation-induced lipid peroxidation as well.

Lipid peroxidatiol! ill biological systellls­Radiati on induced lipid perox idati on has been stud ied in various biological systems as we ll as phospholi pids and liposomes. It is not possible to deal with all a pects of thi s process, Valuable informati on is available elsewhereI5.65.66 and some of the findin gs have been discussed here in the present study. Effect of gamma radiation on an aqueous solution of saturated phospholi pid suggested th at lipid peroxidation could be one of the principal mechanisms of rad iation-induced damage of biological mcmbranes67. Interestingly, a dosc dependent lag pcri od has been reported before the onset of rap iel lipid perox idat ion, on exposure of vesicles enclosed by membranes prepared frolll linole ic acid6R

• However, we have not come across

AGRA WAL & KALE: RADIATIO I DUCED PEROXIDA TIVE DAMAGE: MECHANISM & SIG IFICANCE 295

such a lag period in biological systems. Peroxidation of membranes can lead to the loss of double bonds and breaks in fatty acid side chain in the region of double bond and in turn produce physical changes in the membrane69. Proteins seem to have an important role in determining the radiosensitivity of membranes. Proteins are shown to alter the conformation of both acyl chains and cis C=C bonds in liposomes and these altered states are less sensitive to radiation-induced lipid peroxidation 70. However, lipid peroxidation products interact with proteins 71 ·73 and enzymes 74 leading to inhibition of metabolic pathways75.

Microsomal membrane is a popular model to study lipid peroxidation. The levels of lipid peroxidation products in microsomal membranes prepared from the liver of rat which were irradiated with gamma rays (l Gy) were increased, where as the rates of NADPH­oxidation and NADPH-ferricyanide reduction decreased. TBARS levels and the rate of NADPH­reductase were brought back to the control value 4 days after irradiation 76. Surprisingly, whole body irradiation did not increase lipid peroxidation in microsomes of bone marrow cells, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of bone marrow cell microsomes with 0.1, 10 and 50 Gy caused an increase in lipid peroxidation 77. Further work aimed at understanding this differential response of microsomes may reveal some new facts about the conditions which influence the extent of lipid peroxidation. Alterations in terms of lipid peroxidation, in mice brain synaptosomes have also been seen after whole-body gamma irradiation at 15cGy with a dose rate of 0.01, 0.25 and 9.0 cGy/min)7s. Gamma radiation has been shown to alter the energetics of rat liver mitochondria. Forty eight hours after irradiation, state 3 (V3), state 4(V4) and the rate of A TP synthesis becomes almost normal in spite of accumulation of lipid peroxide products 79. Gamma radiation doses between 1 and 2 Gy induce lipid peroxidation in the nuclear membrane of the liver of pregnant rats and their embryos. Data have also demonstrated high radiosensitivity of the cell nucleus during gestation and embryogenesisso. Enhanced levels of lipid peroxidation have been reported in irradiated erythrocyte ghost membranes81.

Exposure of mice to low dose (15 cGy) of gamma radiation results in an increase in lipid peroxidation in different tissues. In case of varying dose rates (0.0 I, 0.25, and 9 Gy/min), the effect has been found to be

inversely related82.83. These findings are interesting and important, since the range of doses and dose rates used is quite low. However, the data in this range needs to be reexamined and confirmed. Levels of lipid peroxidation are found to increase in blood as a result of whole-body irradiation (0.5 Gy and l.0 Gy) of rats84. Rats on exposure to a total dose of 0.75 Gy of gamma radiation (receiving three doses of 0.25 Gy each at weekly intervals), show a large accumulation of lipid peroxidation products, as conjugated dienes , ketotrienes and TBARS. At the same time, the contents of CoA, pantothenic acid, total phospholipids, total glutathione and GSHlGSSG ratio are considerably decreased, whereas NAD/NADH ratio increases85. Enhanced levels of lipid peroxidation in the liver of rat, induced by gamma­radiation are accompanied by a decrease in the activity of SOD86. Increased SOD activities and decreased lipid peroxide levels induced by low doses of irradiation (0.05-0.5 Gy) have also been reported in the different tissues of ratS7. Decreased levels of specific activities of SOD and GSH-reductase as well as contents of GSH in the erythrocytes of rats irradiated with 8 Gy have been suggested to be the main factor responsible for the increased levels of lipid peroxidation8s. When lipolytic activity and lipid peroxidation after whole body gamma irradiation are examined in rat epididymal adipose tissue, an increase in lipid peroxidation accompanies a decrease in the lipolytic activity89. Decreased lipolysis is likely to be an additional deteriorating factor contributing to radiation-induced death in animals. Decreased levels of Vitamin E content in plasma, brain and liver of rat after whole-body irradiation has been suggested to be responsible for the increased lipid peroxidation in tissues9o. Correlation have been observed between radiation-induced lipid peroxidation and Mg2+_ ATPase activity in thymocytes91. A marked increase in the levels of MT (meta1lothionein) in the brain of transgenic mice has been observed after exposure to radiation (2 to 20 Gy). However, peroxide levels remain unchanged92

• Therefore, it is quite possible that the induced synthesis of MT may be one of the mechanisms that prevents the induction of lipid peroxidation in brain.

Adaptive response has been studied using lipid peroxidation as a biological end point. Pre-exposure of testes to a low dose of heavy ions or gamma rays (0.05 Gy) renders the organ more resistant to subsequent high-dose of irradiation (2 Gy). Increase

296 INDIAN J EXP BIOL, APRIL 2001

in SOD activity and decrease in lipid-peroxidation levels induced by low dose irradiation is suggested to be involved in thi s resi stancc. Adaptive response with heavy ion irradialion is greater than with gamma rays93.

Changes in lipid peroxide levels (TBARS) in serum and heart ti ssue as well as creatine kinase (CPK) activity in serum have been studied as early indicators of peroxidizing effect of heart damage after fract ionated gamma-irradiation (4x5 Gy) . An increase has been observed in TBARS and specific activity of CPK due to the radiati on effect. Application of vitamin E diminishes these levels of TBARS and the acti vity of CPK which is indicati ve of in vo lvement of peroxidation related mechani m in myocardi al dysfunction9~. It is important that reduced rates of lipid peroxidation have been noticed in Yoshida hepatoma cells and microsomes compared to the control (normal rat liver) on expo ure to gamma­radiation . The relative concentrations of alpha­tocopherol and polyunsaturated fa tty acids are shown to be responsible for resistance to lipid peroxidation95. In experiments on Ehrlich ascites tumor cells, it has been reported that ga mma radiation induced lipid peroxidation is accompanied by a decrease in endogenous thiol content'J6.

Interesting findings are avai lable on radi ati on­induced lipid peroxidati ve stress in children coupled wi th a deficit of essential ant ioxidants97. Catabolic products of lipid peroxidati ve ca cade, including diene conjugates, ketobodi es and carbonyl compounds were determined in blood plasma of 428 chi ldrcn aged 0-7 years, residents of area contaminated with radionuclides after Chernobyl Power Plant accident in Russia. Before examination, one multi vitamin therapy was admini stered to a group of children (Group I, n=90) and compared to the group that received no therapy (group II). Group II had 2-6 times higher mean levels of all lipid peroxidative cascade catabolic products and 2-3 times lower levels of vitamins E and A in comparison to age-matched sub-groups of group I. The lipid peroxide cascade system di sorder developed at low doses and di splayed no threshold effect. These findings suggested that these disorders resul ted from free radical chain reaction of lipid peroxidation caused by chronic exposure to low doses of ionizing radiation under conditions of vitamin deficiency.

Oxidation of lipoproteins--Oxidation of lipoproteins has serious implications as oxidatively

altered lipoproteins are suggested to be involved in the development of atherosclerotic process98

.

Radiation effects have been examined in lipoproteins of hamster99, rats 100 and humanI 01.103. Low-density lipoproteins (LDL) have been fou nd to be more susceptible to perox idative damage induced by ionizing radiation . Therefore, ox idized LDL is believed to play a role in events that initi ate atherosc lerosis 102. The greater resistance of lipoprotein(a) has been found to be due to the presence of apolipoprote in (A)I~. Cosmic radiation in space also induces lipid perox idation in human LDL. In contrast, apolipoprotein(B) is not affected by

. d" 101 S' ' fi f' cosmic ra lalion . Igl11 Icant sources 0 space radiation hazards to humans are the trapped radiation belts, solar particle events and galactic cosmic rays 105. Moreover, the exposure of the passengers and crew to background levels on high altitude aircraft are also ex pected to be hi gh and might even be above the recommended maximum acceptable levels l06. Since space travelling is going to be a regular feature sooner than later, there is a need to understand the detrimental effects of cosmic radiation using various biological end points. It will help to assess the risk involved in exposure to cosmic radiat ion.

High LET radialion and lipid peroxidalioll-There is a growing interest in radiobiological studies with high linear energy transfer (LET) radiation due to its application in radiation therapy of cancer and its importance in radiation protec ti on, especially manned space fli ght. Although cellular membranes are vital elements and their integri ty is essential for the viability of cells, meagre informat ion is available about the effects of high LET radi ation on themI 07.IO ' compared to DNA damage lO9. Unli ke DNA molecule, which is a critical target with a small cross-section (3.1xlO-6 11m2), the membranc has a large geometrical cross-section (the di ameter of erythrocytes is 7 11m and microsomes about 0.3 11m). Hence the probability of damage is more due to a direct hit as well as local dose deposition following the definite track structure of emitted delta electrons and the subsequent production of free radicals. In view of th is and new findings in signal transduction as well as in apoptosis, the effect of high-LET radiation on biological membranes is likely to emerge as an important area of research .

Lipid perox idation induced by high-LET radiation has been investigated in liposomes. With increasi ng LET (values between 30 to 15000 KeY/11m) , the

AGRAWAL & KALE: RADIATIO INDUCED PEROXIDATIVE DAMAGE: MECHANISM & SIGNIFICANCE 297

formation of lipid peroxide products such as conjugated dienes, lipid hydroperoxides and TBARS have been found to decrea e llO. Although, the results are just opposite to that found with gamma rays or X­rays, they are not unexpected. The decreased pattern of lipid peroxidation will be discussed in one of the sections. However, fast neutrons with an energy of 1.5-2.0 MeV, increase the rate of lipid peroxidation more markedly than gamma-radiation, in lymphocyte plasma membranes after whole-body irradiation of rats II I.

When microsomes prepared from the liver of mice, are irradiated with 7Li and l(iO having maximum LET values of 354 and 1130 KeV/llm, respectively, lipid peroxidation increases with fluence (8x 1 06 to 3x 1 07

particles/cm2; Ref. 112). Energy deposited by 7Li and 160 ions decomposes water molecules into different free radicals including HO· . Generation of HO· is known to increase in line with the energy deposited by the charged particles. Therefore, enhancement of lipid peroxidation as a function of fluence of 7Li and 160 ions may have resulted from increase in free radical formation and their subsequent reactions with PUFA leading to peroxidative damage. Peroxidative damage is shown to shorten the fatty acyl chain, remove unsaturation and induce cross-linking among the lipid and protein molecules21 . 160 particles have been observed to be more effective than 7Li in inducing microsomal lipid peroxidation, which may be attributed to the difference in ionization densities in the track formed as a result of traversal of particles through microsomal system. Since maximum LET of 160 ions (1130 Ke V 111m), is higher than 7Li ions (354 Ke V 111m), more free radicals are expected to be formed along the track of 160 ions and available for interaction with microsomes to 1l11tlate lipid peroxidation l07. Thus, the biological membranes can be peroxidised on exposure to high LET radiation.

The pattern of lipid peroxidation induced by 7Li and 160 appears to be same as that of gamma radiation. However, the slope of formation of lipid peroxidation with respect to gamma radiation decreases continuously with time, whereas for 7Li and 160 ions, the slope with respect to particle fluence increases at 3 hr and declines gradually thereafter l07. Difference in mode and magnitude of slopes of formation of lipid peroxidation due to gamma radiation and charged particles can be ascribed to different yields of free radicals and molecular products in the respecti ve tracks.

Lipid peroxidation induced by n-y (neutron + gamma) mixed radiation has been studied In microsomes prepared from the liver of mouse irradiated with various doses (2.57 to 17 .07 cGy) . Peroxidative damage increases with radiation dose" 2." 3

. Since it is known that the neutron radiation used in the medical applications is inevitably contaminated with y-rays; the capture of neutrons produces y-radiation in ti ssues; fi s ion neutrons and y radi ations are involved in accidental whole-body exposure and that the natural background radiation to which human beings have been exposed since long, also contain neutrons and y-rays, the above findings may have some significance. It should be noted that the radiation doses used in the studies are quite low. A dose-dependent linear increase of lipid peroxidation has been observed on exposure of dried thin film of lipid to alpha particles" 4. These findings suggest that lipid peroxidation can be induced by different types of particle radiation .

Dose rate of radiation and lipid peroxidalion­Inverse dose rate effect has been proposed as an index of involvement of lipid peroxidation process in radiation induced biological effects 15.11 5. Several reports have convincingly demonstrated that radiation

. d 16.22. 11 5·120 effects are Inversely related to the ose-rate . . Our results have shown that radiation induced lipid peroxidation increases with a decrease in dose rateI6.121 . Decreased radiation effect observed at higher dose rate is probably because of greater recombination of extremely short lived and highly reactive primary free radicals. Chances of indirect radiation damage increase when free radicals react with lipid molecules without recombination as it readily happens when the dose rate is decreased. Therefore, more biological damage is expected in lower dose-rate than a higher one.

Lipid peroxidation and apoptosis--Radiation induced cell death has classically been defined as either interphase or reproductive cell death 122. Mitotic failure is known to result in reproductive cell death . Interphase death, defined as metabolic cell death occurs prior to the first cell division. Distincti ve characteristics like cell shrinkage, membrane blebbing, chromatin condensation, D A fragmentation and ultimate fragmentation of the cell into membrane enclosed vesicles designated as apoptotic bodies, associated with interphase cell death 123

·124 have been termed as apoptosis9

. Apoptosis is a highly organized physiologic mechani sm of

298 INDIAN J EXP BIOL, APRIL 2001

destroying injured and abnormal cells. The plasma membrane is suggested to play an important role in initiation of apoptosis36. As mentioned earlier, lipid peroxidation is an important effect of radiation on membranes. Various studies have linked radiation, lipid peroxidation-induced alterations and/or

. 125·144 Th I f d' . h apoptosls . e resu ts rom stu les Wit liposomesl45, red cell ghost membranes l46 and cells in culturel47, murine thymocytes l48, MOLT-4 human leukemic cells l28 and 234 murine T hybridomas l25

suggested that membrane lipid peroxidation is an early biochemical event in irradiated cells that might contribute to apoptotic signaling. Importantly, lipid peroxidation occurs much earlier than DNA fragmentation and loss of cell viabili ty l25. These relatively new findings also provide evidence to the idea that biological membranes are also critical targets for radiation. The fact that membrane lipid peroxidation could be a critical initial event that can participate in the induction of apoptosis has been supported by observations with membrane protecting

. 'd d 149-156 Th I I' k b antioxi ant compoun s . e c ose In etween lipid peroxidation and apoptosis is likely to open new horizons of radiation research. A growing interest in radiation-induced peroxidative damage, signal transduction and cell death is already visible. Gamma irradiation has been shown to activate protein kinase C (PKC) and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes 157. Recent studies have suggested that radiation causes activation of a phophatidylinositol-specific phospholipase C (PI­PLC) through hydroxyl radical generation, leading to diacylglycerol production and in turn PKC activation 157. Borges and Linden 158 have found that irradiation leads to two waves of apoptosis in distinct cell populations of retina of new born rats. Pyrrolidine thiocarbamate, which inhibits lipid peroxidation, prevents the early wave of apoptosis.

Lipid peroxidation-Non-linear pattern. concen­tration of lipids. kinetics alld thermodynamics­Although membranes are vital for cell function, their role as critical targets of radiation has not been given enough attention. On the other hand, efforts have been made to prove that membrane damage is not a critical event or has no significant contribution. Despite these facts attempts have been made to understand the radiation-induced membrane damage in terms of lipid pcroxidation. Some of the results arc discussed earlier. In the present section, we have examined a

few interesting aspects such as the effect of lipid concentration on peroxidation itself, its non-linear pattern, kinetics and thermodynamics.

Extent of lipid peroxidation depends on the dose of radiation. To study the effect of dose, we have mainly used gamma-rays to induce lipid peroxidation in liposomes, microsomes and ghost membranes prepared from erythrocytes. Interestingly, the concentrations of microsomes and ghost membranes influence the lipid peroxidation. When different concentrations (mg protein/mL) of' microsomes and ghost membranes are irradiated, the lipid peroxidation decreases with their concentration 121.1 59. Higher level of lipid peroxidation at lower concentrations have been ascribed to dispersal of reticulum in aqueous phase, or a change in concentration of heme proteins in preparations 160. However, we have provided the alternative explanation . Lipid perox idation occurs mainly through formation of free radicals . When different concentrations of ghost membranes and microsomes are irradiated with a constant dose, extent of lipid peroxidation is likely to be the same, as the free radicals generated at each concentration of ghost membranes and microsomes are expected to be almost the same in number. Estimation of lipid peroxidation per mg protein may therefore lead to decrease in its level with an increase in protein concentration 121. These results have been confirmed by other workers 161. It is important that the extent of lipid peroxidation and the concentration of membranes should not be ignored, otherwise it may lead to wrong conclusion. In case of liposomes, lipid peroxidation increases linearly with an increase in the concentration of liposomel6

.

Since lipid peroxidation is detrimental to aerobic life, research into this process has been quite intense during last several years. Lipid peroxidation involving hepatic microsomal membranes is of great importance and is considered to be a valuable tool in the study of oxidative stress l62 . NADPH, ascorbate or ferrous ions have been used to initiate microsomal lipid peroxidation. Therefore, the effect of their concentration on peroxidation has also been studied. These factors (NADPH, ascorbate and ferrous ions) enhance lipid peroxidation in a concentration dependent manner except in the case of higher concentration of ferrous ions. At higher concentration of ferrous ions, lipid peroxidation c1eclines I63 . At relatively higher concentrations, iron has been suggested to behave as a dominant chain-terminating

AGRA WAL & KALE: RADIATIO INDUCED PEROXIDATIVE DAMAGE: MECHANISM & SIG IFICANCE 299

antioxidant62 and may be responsible for these lower levels of lipid peroxidation.

On examining the kinetics and thermodynamics of NADPH-dependent microsomal lipid peroxidation '64

apparent Km and Ymax increase with temperature and can be linked to enzyme substrate affinity and the turnover number of enzymes. Since, lipid peroxidation is a free radical process involving a sequence of reactions, Km can be related to the rate constants of these individual steps . Moreover, the linear dependence of Ymax on temperature suggests that the velocity be controlled mainly by composite

rate constant. Change in the enthalpy (~H) and entropy (~S) of lipid peroxidation process is positive

whereas the change in free energy (~G) is negative. It seems that NADPH-dependent lipid peroxidation is mainly an entropy driven process which is opposed by unfavourable change in enthalpy .

Liposomes prepared from L-u-lecithin exhibit non­linear pattern of radiation-induced lipid peroxidation . When liposomes are irradiated with different doses of radiation, lipid peroxidation increases with dose upto 330 Gy (lower dose region) and decreases with dose beyond 330 Gy (higher dose region). This suggests that in the low dose region most of the energy deposited into liposomes is translated into damage. On the other hand, energy deposited in the higher dose region may not have completely translated into damage and some part of it might have dissipated towards the recombination processes of free radicals leading to formation of relatively stable and unreactive molecular products. Decrease in lipid peroxidation may be due to avoidance of the propagation stepl6.

It is known that vitamin E scavenges oxygen free radicals and suppresses free radical chain reaction, Fe2

+ ions decompose radiolytically formed molecules like H20 2 and organic peroxides (LOOH), and molecular oxygen propagates lipid peroxidation. However, these chemicals do not alter the non-linear relationship between radiation dose and lipid peroxidation of Liposomes' 63 . This non-linear pattern . cd· h 159 d· I IS not loun In eryt rocytes an mlcrosoma systeml21. Lipid peroxidation increases with increase in radiation dose. This linear pattern in both the systems may have arisen due to the presence of various factors involved either in initiation or propagation of lipid peroxidation. Ferritin is ab le to enhance the lipid peroxidation and change the non­linear pattern into a linear one. However, lactoferrin

and transferrin fai l to do so (Kale and Sitasawad, unpublished).

Inhibition of lipid peroxidation-Being a free radical chain reaction, lipid peroxidation causes membrane damage as well as oxidative modification of critical targets. Therefore chemical agents, which can interact with these free radicals and scavenge them would be effective in inhibiting lipid peroxidation and in turn protect against radiation damage. Large number of chemicals have been tested for this ability. However, the findings with some agents only will be discussed here.

Recently, nitroxides as a new class of radioprotectors have been identified' 65.'67. Nitroxides have been shown to react with a variety of biological oxidants including oxygen free radicals 168-172. Most of the nitroxides are membrane permeable. They exert their action intracellularlyI 65.173. Therefore, nitroxides are also expected to be effective inhibitors of oxidative damage. When liposomes are irradiated in presence of tempo and tempol , both the nitroxides inhibit oxidative damage '74 . In an another study, tempo and tempol protect the acyl chains of EPCSUY (egg phosphatidylcholine small unilamellar vesicles), including the highly sensitive polyunsaturated acyl ehainl74. Interestingly, these nitroxides protect via a catalytic mode. Nitroxide radicals can be restored and terminate free radical chain reaction in a catalytic manner. Nitroxide neither yields secondary radical upon their interaction with oxygen free radical nor act as oxidants. Importantly, not only are nitroxides self­replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers very effective strategy to protect liposomes, biological membranes and other lipid based assemblies from radiation damage.

There has been an increasing interest in modifying

properties of micronutrients like vitamin E (u­tocopherol) against oxidative stress/damagel75. Vitamin E is naturally present in mammalian cell membrane and is known as a micromembrane stabilizer'76. It protects against cell membrane damage from lipid peroxidation 177

. Clinical use of vitamin E has been shown to be, even at an oral dose, as high as 3200 mg/day l78. Lipid peroxidation induced by gamma radiation in linoleic acid emulsion is inhibited by vitamin E179. As mentioned earlier, changes in lipid peroxide levels (TBARS) in rat serum and heart tissue as well as creatine kinase (CPK) activity in serum have been examined as early indicators of

300 INDIAN 1 EXP BIOL, APRIL 2001

peroxidising effects of heart damage after fractionated gamma-irradiation. Application of vitamin E diminished these levels of TBARS in serum and in heart homogenates and also CPK activity in serum l80. Lipid peroxidation induced in plasma, brain and liver after whole body irradiation of rat is inhibited by administration of Vitamin E89. It may be noted that vitamin E is a chain breaking antioxidant and stoichiometrically inhibits lipid peroxidation.

Trolox, a water soluble analog of vitamin E, as expected, is an effective inhibitor of membrane lipid peroxidationI49- 153. Trolox, also a chain breaking antioxidant, penetrates biomembranes more rapidly than vitamin E. The observation that early treatment with trolox inhibits apoptosis, suggests that membrane lipid peroxidation is a critical event in the series of steps that lead to apoptosisl 54

• Dihydrolipoic acid and N-(2-mercaptoethyl)-1,3-propane diamine are also inhibitors of membrane lipid peroxidation and show similar results as that of trolox 155.156. Ebselen, a selenoorganic compound that exhibits glutathione peroxidase-like activity similar to that of phospholipid hydroperoxide glutathione peroxidase, is shown to inhibit radiation induced apoptosis in thymocytes by reducing peroxides formed during and after radiation l48 . An excellent account on trolox and related compounds is available elsewhere36.

Ever since Patt et aL. and Bacq et aL. have shown radioprotection by cysteine l81 and its carboxylated derivative cysteamine l82 against the lethal effect of X­rays, numerous thiol compounds have been tested for their ability as radioprotectors and for their usefulness in radiation therapy of cancerI 83-185. Thiols have been widely studied 186-189 as radioprotective agents using various biological end points. The protective action of thiols is ascribed to scavenging of free radicals, restoration of damaged target molecules by H· or electron (e-) transfer; induction of hypoxia, chelation of metal ions and formation of mixed disulfides. Thiol compounds render protection against radiation­induced lipid peroxidation 101.190-192 probably following the aforementioned mechanistic pathways.

Study on plants as modifiers of radiation effects is a new area of research. Many phytochemicals from plants are known to possess antioxidant properties. Indeed, human beings are consuming a variety of antioxidants in their diet and are thus protected from radiation exposure. Therefore, it is necessary to assess the protective action of such commonly used phytochemicals and exploit their possible application

in radiation therapy of cancer. Phytochemicals are likely to serve as an alternative source of non-toxic radioprotectors . In view of this, it is important to note that plant extracts and phytochemicals inhibit radiation-induced lipid peroxidation in model as well as in animal systems90.107.179.180.193-195 .. Further work

with phytochemicals and plant extracts needs to be taken up because a large number of chemical agents like thiols and other compounds have been tested for their protective property, but most of them have not been accepted clinically due to their toxicity.

Other chemical agents are also reported to inhibit radiation-induced lipid peroxidation . Propofol has a protective effect against peroxidation in linoleic acid emulsion and suggested that it could be beneficial as an anaesthetic or sedative drug in patients presenting pathologies associated with free radicals l79. Antioxidant effect of probucol , an antiatherogenic and anti atherosclerotic drug, has been evaluated on peroxidation of low-density lipoproteins (LDLs). It decreases the yields of TBARS and conjugated dienes. However, probucol is non-reactive with peroxyl radicals 103, which are known to be involved in propagation of lipid peroxidation. Probucol is also known to modulate lipid peroxidation in human LDL induced by environment in space and have a protective effect on peroxidative stress 101 . Since oxidized LDL and decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, it is an important finding that DHEA is able to inhibit the oxidation of LDLs obtained from healthy subjects, in terms of conjugated dienes and TBARS formation, as well as by reducing vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes l02 . Antioxidant properties of radioprotectors have also been examined in different tissues of animals like mice and rats . Pantothenol 193, phenoxane82, zinc metallothionein 196, dipyridamole J97

and succinate l98 have been shown to inhibit lipid peroxidation in the liver of rats, blood and tissue of mice, rodent bone marrow cells, liver of spleen of mice and in mitochondria respecti vely.

Although radioprotectors have contributed , to a great extent, in understanding the radiation effects, they have not been accepted for practical applications . A survey of literature suggests that without addressing or understanding the non-acceptabili ty of several protectors, further work on radioprotectors is either abandoned or diverted towards the search and testing

AGRA WAL & KALE: RADIATION I DUCED PEROXIDATIVE DAMAGE: MECHANISM & SIGNIFICANCE 30 I

of new agents. This kind of endeavour has helped very little, if at all, to radiation protection or radiation therapy. Therefore, the gap between the present state of knowledge and radi ation protection/radiation therapy of cancer remains to be bridged.

Modulation of lipid peroxidation by calciulI! and phellothiazines-A chemical or drug, which enhances the formation of free radicals, involved in initiation and/or propagation can increase the extent of lipid peroxidation. On the other hand, the removal of these free radicals can lead to inhibition of lipid peroxidation. Therefore, we have extensively used the lipid peroxidation process to understand the effect of phenothiazinesl fi.J7. 121.1 9lJ, 2-mercaptopropionyl gly­cinel59, vitamin E, ferrous ions and oxygenl63 , calcium ions 159.164.200.202, catalase l6, divalent transition metal ions203 and extract of some important plants. However in the present communication, only the results with calcium ions and phenothiazines have been discussed in brief to show the usefulness of the peroxidation process.

Modulatory effect of Ca2+ on lipid peroxidation is a controversial subject, but both lipid peroxidation and Ca2+ are important for a biological system. We have examined the influence of Ca2+ on lipid peroxidation in microsomes prepared from liver of mice, initi ated by ADPH, ascorbic acid and ferrous ions. Ca2+ modulates lipid peroxidation in all these three systems. Mode and magnitude depend on' the system and the concentration of cofactors used for the initiation of lipid peroxidation and not only on the concentration of Ca2+. In the ascorbate system, Ca2+ enhances lipid peroxidation upto 30 j..lM concentration of ascorbic acid and inhibits the same beyond 30 j..lM. It increases NADPH-dependent lipid peroxidation at all concentrations. Depending on the concentration of ferrous ions, lipid peroxidation decreases or increases in the presence of Ca2+ ions (Ref. 200). Thus, these findings reveal the reason of controversial reports. These findings also reveal that the protective effect of Ca2+ is not an artifact. We have argued that the explanations provided earlier for the enhancement as well as inhibition of lipid peroxidation by Ca2+ are speculative in nature and some of the basic facts of chemistry have been overlooked. Since Ca2+ is a non­variable valence state ion, it cannot participate directly in free radical reactions of lipid peroxidation. Therefore, it is suggested that Ca2+ may modulate lipid peroxidation through some biochemical processes or its interaction with membranes.

Effect of Ca2+ on kinetics and thermodynamics of ADPH-dependent microsomal lipid peroxidation in

microsomes prepared from the liver of Swiss albino mice has been studied 159. Ca2+ increases the Vmax but not the Km. Ca2+ perhaps enhances the velocity of the reaction and does not affect enzyme-substrate affinity. Results also show non-competiti ve and mixed type of activation with Ca2+. The energy of activation (Ea) decreases in presence of Ca2+. It is important to note that Ca2+ ions can modulate L\H , L\S and L\G of the lipid peroxidation process. The value of L\H decreases in the presence of Ca2+, which is an indicative of a requirement of low heat of activat ion in the formation and breakdown of iron-oxygen complex in transition state. Since Ca2+ enhances NADPH-dependent lipid peroxidation200, change in entropy is expected to be more posi ti ve in presence of Ca2+ due to di sturbance in the membrane organization. On the contrary, Ca2+ lowers the L\S of lipid peroxidation. It is possible that with increase in lipid peroxidation in the presence of Ca2+, there is a lower level of unsaturation in the structure of PUF A and perhaps an enhanced cross­linking among the molecules, thus increasing the rigidi ty of biomembranes which results in a low value of L\S . Although L\G of lipid peroxidation increases in the presence of Ca2+, the overall change is negative and favourable. Our findings are significant from the pathophysiological point of view. Although Ca"+ regulates a variety of biochemical and cellular functions, increase in its concentration above the physiological level can make the cellular environment thermodynamically and/or kinetically favourable for reactions leading to membrane damage and cell death.

Since Ca2+ is involved in cell damage and death, the integrity of membranes is essential to maintain Ca2+ homeostasis. In radiolytic systems, as mentioned earlier, membranes are critical targets of detrimental effects. Therefore, damage and death due to Ca2+ as well as ionizing radiation seems to be interlinked. Hence, the possible interactions of Ca2+with radiation induced damage have been examined using the lipid peroxidation process . Importantly, Ca2+ inhibits lipid peroxidation in microsomes and ghost membranes prepared from erythrocytes 159.201.204. The inhibitory effect in presence of calcium and calmodulin confirms the involvement of Ca2+ in peroxidative process. Protective effect of Ca2+ has been ascribed to the removal of peroxidised fatty acids from the membrane by Ca2+dependent enzyme phospholipase A2 and subsequent disposal of peroxides 199. A23187, an

302 INDIAN J EXP BIOL, APRIL 200 1

ionophore specific for Ca2+, decreases the protective effect of exogenously added Ca2+. This suggests that Ca2+ protects the cell from the extracellu lar side against peroxidative damage. It is therefore speculated, that extracellular concentration of Ca2+, may play an important role in determining the radiosensitivity of the cell. To find out the significance and relevance of these observations, we have studied the modifying effect of Ca2+ on the survival of E. coli 8000. Interestingly , pre-irradiation treatment of Ca2+enhances the bacteri al survival and on the other hand, post-in·adiation treatment increases the killing of bacteria204

. Pretreatment of Ca2+ may increase the stabi lity in membranes and give protection to E. coli against rad iation effect. On the contrary, post-irradiation treatment of Ca2+ may cause more killing because these cells probably did not maintain Ca2+ homeostas is due to damage to their membrane II 8.205. Accumulation of Ca2+ inside the cell above a certain level may activate various digestive enzymes resulting into cell death. Thus, lipid peroxidation is useful to understand the mechanistic aspects and significance of Ca2+ in radioprotection.

Radiation therapy is one of the most important and popular tools for cancer treatment. A relatively small portion of hypoxic cells are present in tumors. Since hypoxia has a protective effect against ionizing radiation, the presence of hypoxic cells in tumors limits the success of radiation therapy of cancer. The possibility of use of radiomodifiers which could either selectively potentiate the radiation effect in tumors (particularly in hypoxic cells) or conversely protect surrounding normal tissue opened newer avenues in the radiation therapy of cancer. Numerous chemical agents have been tested for their ability to potentiate

. d·· ff 65206·208 S f or protect agall1st ra latlon e ect · . orne 0

them like phenothiazines (PZ) have shown promising results. These drugs selectively kill hypoxic cells in

d I · 209·2 14 H tumors an protect norma tissue . owever, mechanism of this dual effect has not been well understood. Therefore, an attempt has been made to understand this duality using the lipid peroxidation process. PZ inhibit radiation-induced lipid peroxidation 12 1. Interestingly, the protec ti ve effect of PZ against lipid peroxidation diminishes in the presence of Fe2+ ions. On the other hand, the protecti ve effect enhances in the presence of Fe3+ ions l21

. Similar results have been obtai ned with the I I 2 15 d . I . I d· '16 g yoxa ase sys tem an al1lma survlva stu les- .

On the basis of these and other related findi ngs it has

been hypothesized that differential radiosensitization of tumor cells and radioprotection of normal cells by phenothiazines is related to the presence of Fe2+ and Fe3+ ions respectively2I7. As a re ult of changed biochemical environment such as pH, hypoxi a and accumulation of ferritin, Fe2+ ion concentration in tumors especially in hypoxic cells is expected to be higher (which is further enhanced by radiation) than in the well oxygenated normal cells. In normal cells, iron is in the Fe3+ form predominantly, which may be responsible for the protective effect of phenothiazines. Fe2+ ions increase damage by various oxidative reactions. Autooxidation of Fe2+ is contingent on availability of chelators that lower the reduction potential of Fe2+/Fe3+ couple, making one electron transfer from Fe2+ thermodynamically more feasible2I8

. Therefore, the redox activity of iron may be altered by phenothiazines or vice versa in such a way that their effect on radiation-induced changes is enhanced in the presence of Fe2+ ions and inhibited in the presence of Fe3+ ions215 . This possibility has been tested by using naturally occurri ng hydroxy naphthoquinones and their ion complexes as modulators of radiation-induced lipid peroxidation in synaptosomes2 19 as well as metal-dafonates which show antioxidant and proxidant effect on NADPH­dependent lipid peroxidation in murine hepatic microsomal system220

. Thus, lipid peroxidation process is useful to explain the dual effect of phenothiazines against radiation damage. Our findings with phenothiazines show that hypoxia in tumors may not be a limiting factor and may be exploited for radiation therapy of cancer by employing suitable metal-based chemomodulation.

Conclusion and perspective Lipid peroxidation being a highly destructive

process, brings about various changes in biological membranes causing cell damage and death. A free radical which has the ability to ab tract hydrogen homolytically from the methylene carbon of PUFA can initiate and molecular oxygen can propagate lipid peroxidation through formation of peroxyl radical. Rate of initiation is always faster than propagation. Lipid peroxidation has no intrinsic metal ion requirement, but if present can initiate and/or propagate the process. Hi gher concentrations of Fe2+ ions may have an inhibitory effect. Radiation-induced lipid peroxidation has been studi ed in various biological sys tems as well as phospholipids and

AGRA WAL & KALE: RADIATIO INDUCED PEROXIDATIVE DAMAGE: MECHANISM & SIG IFICANCE 303

liposomes. Biological membranes have been found to be peroxidised on exposure to high LET radiation. Pattern of lipid peroxidation induced by high LET is almost the same as that of gamma radiation. The extent of radiation-induced lipid peroxidation depends on the dose and dose rate of ionizing radiation. Several chemical agents have been tested for their radioprotective ability and their usefulness with the help of the lipid peroxidation process. In non­radiolytic systems, lipid peroxidation is increased with an increase in the concentration of NADPH, ferrous ions (except in higher concentrations) and ascorbate. Lipid peroxidation process is quite useful for modulation studies and explains the paradoxical role of Ca2

+ on peroxidative process as well as the dual action of phenothiazines against radiation damage. Results with phenothiazines suggest the possibility to exploit hypoxia for the radiation therapy of cancer.

Known chemical aspect of lipid peroxidation have not been well uti Ii zed for radiobiological studies due to non-acceptance of membrane damage as a cri tical event in determining the detrimental effects of ionizing radiation in biological systems. Recently plasma membrane has been shown to play an important role in initiation of apoptotic cell death and that lipid peroxidation of the plasma membrane is an early biochemical event in irradiated cells which contributes to apoptoti c signaling. The close link between lipid pelOxidation and apoptosis is expected to open new horizons for radiation research. Work needs to be focussed on biochemical and molecular pathways involved in lipid peroxidation mediated apoptosis to understand the ri k as well as benefit of ionizing radiation.

Acknowledgement Authors would like to acknowledge the financial

support provided by UGC, CSIR, ICMR and The Nuclear Science Centre, New Delhi , in the form of research grant and fe llowship.

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