Indian Journal of Experimental Biology Vol. 40, June 2002, pp. 680-692

Biological significance of singlet oxygen

Thomas P A Devasagayam & Jayashree P Kamat

Cell Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, Indi a

Phone: 022-5593948; Fax.: 022-5505151/5519613; email: tpad@apsara.barc.ernet.in

The biological significance of singlet oxygen (1 0 2), an electronically excited species of oxygen , has been realized only in the last two decades. This was mainly due to the lack of proper methodology to generate this reactive oxygen species (ROS) in pure form and its reactions with biological molecules. Recent studies, using new ly developed detection methods, show that 102 being generated in many biological systems, can sign ificantly and quite often adversely alter several crucial biomolecules including DNA, proteins and lipids with undesirable consequences including cytotoxicity andlor disesase de­velopment. The reactions of 102 with the biological molecules are rather specific, as compared to other ROS . There are vari­ous compounds, mainly derived from natural sources that offer protection against damage induced by 102, Among the ant i­ox idants carotenoids are the most effective singlet oxygen quenchers followed by tocopherol s and others. The same reactive spec ies if generated specifically in diseased states such as cancer can lead to the cure of the disease, and this principle is utili zed in the newly developing modality of cancer treatment name ly photodynamic therapy. Singlet oxygen, in low con­centrations can also act as signaling molecule with several biological implications. T his review clearly brings out the bio­logical significance of 102,

People have become more health conscious in recent years. One etiologic agent implicated in diseased state is 'oxidative stress' which involves excess generation of prooxidants. These species, in biological systems include excited states, free radicals and other related species mainly derived from oxygen and nitrogen. As such, prooxidants are generated in our body during the normal metabolic processes as well as during ex­posure to adverse pathophysiological conditions. In a healthy human body the generation of prooxidants in the form of reactive oxygen species (ROS) and reac­tive nitrogen species (RNS) are delicately balanced by the antioxidant defenses. Exposure to prooxidants results in oxidative stress that shifts the balance in favo ur of prooxidantst. ROS of interest, generated during oxidative stress include hydroxyl radical, su­peroxide, peroxyl radical, hydrogen peroxide and singlet oxygen (102), The significance of 102 has been realized only recently due to the development of methods for its generation, free from other contami­nants as well as its detection. Singlet oxygen has been considered as a major cytotoxic species to eukaryotic cells, bacteria and viruses. Extra-cellularly generated 10 2 has been found to be genotoxic to mammalian cells grown in culture. On several instances, singlet oxygen has been implicated in the induction of tu­mour by photosensitization and in the metabolic acti­vation of carcinogens. Besides, it has been implicated in several pathological processes like lung-oxidant injury, skin photosensitivity and erythropoetic por-

phyri a. The latter condition has been shown to be due to accumulation of specific pigments below the skin due to a metabolic defect. Some reports also show that 102 playa significant role in the inactivation of cells or cellular components due to UV -A and near­visible radiation 1-8 . This brief review gives a bird's eye view of the developments in the study of singlet oxygen.

Historical Even though oxygen has undergone two centuries

of investigation, 102 has been recogn ized to exist only from 1924. Starting with its accidental discovery by Howard Seliger2 in 1960, due to its 'glowing ability in the dark', this excited species has been a scientific enigma. During the initial 'astrophysical period' (until 1963) 102 was regarded as a rare spec ies largely of importance in atmospheric physics. The terrestrial significance or its chemical role becomes recognized in the followin g 'chemical period' during which there was an exponential growth of published literature on this reactive species mainly on its chemical nature and reactions. In the last 3 decades, however, scientists from various disciplines like Physic, Chemistry, Bi­ology and Medicine are attracted towards this enig­matic species and have contributed significantl/ to the present-day knowledge about '0 2. Like many other reactive species, this can be harmful at higher concentrations and at low levels may act as signaling molecule.

DEY ASAGA Y AM & KAMAT: SINGLET OXYGEN AND ITS BIOLOGICAL EFFECTS 681

Chemical nature Molecular ground state oxygen, as present in the

biological milieu, is kinetically inert. This nature can be explained by its electronic structure. The two un­paired electrons in the outermost orbit have the same quantum number imposing a spin restriction on the reactivity of oxygen. This , however, can be removed by moving one of the unpaired electrons in a way that alleviates the spin restriction. This phenomenon re­quires an input of energy and generates the singlet states of oxygen. The singlet states of oxygen do not have unpaired electrons and hence do not qualify as a radical. Delta singlet oxygen (I ~g O2) is the most im­portant in biological systems and has 22.5 kcallmole of energy above the ground state. Sigma singlet oxy­gen (ILg O2) has 37.5 kcallmole of energy above the ground state and usually decays to the 1 ~g state before it reacts with another matter (half life in aqueous sys­tem 1O.9s as compared to 1O.6s for the I~g O2). Due to their emissions at specific wavelengths, these species can be detected using different photo-detectors2.8.

Generation in biological systems Singlet oxygen can be generated in biological sys­

tems by two different routes,-by 'light reactions'

due to photo-excitation and by 'dark reactions ' due to chemi-excitation. A major route for the former proc­ess is by the type II photosensitization reaction result­ing in an energy transfer from triplet state of photo­sensitizer to ground state molecular oxygenl .IO.1 2 (see Fig. 1 for various ways for the generation of 102),

Many cellular constituents such as flavins, por­phyrins, cytochromes, 4-thiouridine etc. as well as

ENZYMES

RCOO . + RCOO. O, +3SENSITIZER

~ :~tO ;( H,o,+OCI-'"'''''''''' ~~ \" 1;':1 prod~cts ~ / H,~+CI-

.;A >" '0 ..... I----~-- H,O,+O,'

:: c=~ /y 2~~ o,+::c=o" /H,O, /

Y e OH, + 0;',

+ ZH

OZONIDES

Fig. 1-Ways, by which singlet oxygen can be generated

Table 1-Ways by which singlet oxygen can be detected

S. No. Method

I. By chemiluminescence from radiative transition of 102 to the ground state a. Dimol emission 2'02 ~ 302 + hv (634, 703 nm)

Detection by red sensitive, thermoelectrically cooled photomultiplier b. Monomol emission

102 ~ 30 2 + hv (1270 nm) Detection by germanium diode detector

2. Chemical traps a) Dields-Alder reaction of dienes to form endoperoxides b) 'Ene ' reactions of alkenes to give allylie hydroperoxides c) With alkanes to form 1-2 - dioxetanes (2 + 2 eycloaddition) d) With sulphides to form sulfoxides e) With electron rich phenols to form hydroperoxidienones f) With deoxyguanosine to form endoperox ide g) With lertiary amines to form nitroxyl radical, detectable by ES R h) Histidine ~ endoperoxide formation and oxidation in the presence of nitrosodi methylaniline i) With cholestrol to form 5 a-hydroperoxide derivative

3. Quenchers a) By energy transfer - carotenoids and nickel complexes with high rate constants (- 1010 M·1s· ' ) b) Electron transfer - DABCO Diazabicyelo [2, 2, 2] octane, phenols, sulphides & azides. Lower rate constants

(- 107 to 108 M·1s· ' )

4. Use of dueterated solvent Tn D10 life time 15 -18 times longer than in H20. Same with deuterated organic solvents

Ref. 8

Comments

Sensitivity low

Sensitive Expensive

682 INDIAN J EXP BIOL, JUNE 2002

biologically active drugs like tetracycline, chlorpro­mazine, merbromin, thiazides, psoralens, photosensi ­tizers used in photodynamic therapy (PDT), sus­pended particles in the polluted atmospheres and fullerenes such as C60 and C70 have the abi lity to gen­erate '02 under illumination (Table 1)'3,38. During photosensitization these compounds absorb energy, undergo intersystem crossi ng and transfer energy to the molecular ground state oxygen generating '02.

Dyes such as methy lene blue, rose bengal etc . also generate '02 on photo-excitation.

The dark reactions that generate '02 include enzy­matic reactions catalyzed by dioxygenases, lactoper­oxidases, myeloperoxidases, cytochromes, tryptophan pyrrolase and lipoxygenases. There are evidences for '02 production obtained using a number of purified enzyme systems8

.' 2. Cadenas et a/. 39 obtained evi­dence for '02 participation in the metabolism of ara­chidonic acid by prostaglandin-endoperoxidase syn­thase through the chemi luminescence spectrum at 634 and 703 nm. A simi lar identical spectrum was ob­tained using isolated cytochrome P450 or microsomal fraction s supplemented with hydroperoxide4o. Lipid peroxidation in microsomal fractions initiated by hydroperoxides or iron/ascorbate, as well as in iso­lated hepatocytes has been studied and the available evidence point to the '02 formation via the Russel mechan ism. Singlet oxygen is generated either at the

catalytic site of the enzymes or prod ced by the de­composition of unstable primary oxidation products. It can be produced from peroxyl radicals by Russel mechanism hydrogen peroxide plus hypochlorite, non-enzymatic dismutation of superoxide or dismuta­tion of 1,2-dioxetanes via triplet exci ted ketones.

Metabolic generation of '02 has been shown to oc­cur in stimulated neutrophils. Some 'tudies show that '02 can be derived from the spontaneous rather than enzymatic decomposition of superoxidc. Hence su­peroxide dismutase indirectly protects from '02. This species has also been found to be generated from other react ions of biological relevance such as lipid peroxidation and reaction of hydroperoxides with peroxynitrite.

The formation of '02 by these mechanisms is likely to be increased under the influence of certain xenobi­otics capable of inducing oxidati ve stress. In the mammalian tissues, one of the main candidates for '02 production is the activated polymorphonuclear leukocytes. The primary function of these cells is to destroy invading microbes. In response to such stim­uli, the cells generate superoxide, hydrogen peroxide and hypohalous acids during a process known as 'res­piratory burst' . Fairly large and toxic quantities of '02

are produced during respiratory burst via reactions catalyzed by the lysosomal myeloperoxidase. The generation of '02 by polymorphonuclear neutrophils

Category

Table 2-Chemicals that generate 102 on photoexcitation

Chemicals/drugs Refs.

Cellular constituents Drugs

Photosensitizers for PDT

Plant and bacterial pigments

Other xenobiotics

flavins, porphyrins, cytochromes, 4-thiouridine tetracycl ine, chlorpromazine, merbromin, thiazides, quinine, chloroquine, primaquine, quinacri ne, mefloquine

haematoporphyrin, haematoporphyrin derivative, chlorins, bacteriochlorins, phthalocyanines, hypocrellins, hypericin , meso tetrakis( 4-(carboxymethyleneoxy) phenyl)porphyrin, meta (tetrahydroxyphenyl )chlorin , lutetium bis­ethyltetraazaporphyrin, Merocyanine-540, tin ethyl etiopurpurin-I, tin octaethylbenzochlorin, nitrophenyl ether, 5,1 O, 15,20-tetrakis(4-N-Methylpyridyl)porphyrin 5,10,15,20-tetraarylethynylporphyrinatozincll thiopyrylium, selenopyrylium, telluro­pyrylium

thespone, thespesone, mansonone-D, mansonone-H, anthraquinone, barleriaquinone-I

naphazoiine, si lica, diperoxovanadate, fullerenes. titanium dioxide, p-phenilene vinelene, biphenyl derivatives, azo-dye Orange II , rose Bengal, alloxazines, isoalloxazi nes

2 13 14

15·17 18-19 20 21 22,23 24 25 26 27 28 19

29 30,31

32,33 34,35 36,37

DEY ASAGA Y AM & KAMAT: SINGLET OXYGEN AND ITS BIOLOGICAL EFFECTS 683

or myeloperoxidase during bactericidal ac tion, photo­sensitization and during lactoperox idase activity was proved3

.12.4 1. This provided evidence showing micro­

bacteric idal activity is related to 102 generation . Hu­man saliva, in presence of low amounts of hydrogen peroxide can also generate 102 (ref. 12).

Detection and quantification of t02 Several techniques have been developed for the de­

tection and quantification of 102 (Table 2)8. Among these techniques the highly specific ones are expen­sive. The more practical ones are the ones that use 'traps' and measurement of specific products forma­tion. A variety of spectroscopic techniques have been developed to evaluate the t02-quenching capacity of antioxidants. Infra-red photoemission accompanies the spontaneous decay of 102 quenching. A group of quenching assays is based on a pulse of t02 generated with a photochemical source. The lifetime of the photoemission in absence and presence of carotenoid is measured by time-resolved spectroscopy. The gen­eration of 102 can also be monitored continuously either chemkally or photochemically and the effect of antioxidant on the steady state level of photoemission is evaluated8.41 .42.

Reaction with biological molecules Due to its relatively long half-life (in the range of

\-50 Ils in aqueous systems), t02 can travel apprecia­ble distances in the cellular environment and is capa­ble of damaging various biomolecules2.8. In human plasma, which is rich in antioxidants, the life-time of 102 is calculated to be 1 Ils . It can move freely across water-lipid interfaces. It can behave like a strong elec­trophile in solution and reacts with biomolecules pos­sessing regions of high electron density (for instance guanine in DNA). Oxidative damage in biomolecules mediated by 102 is rather frequent. Lipids, proteins and DNA are all at risk.

Lipids Cellular biomolecules like lipids are the most sus­

ceptible to oxidative damage. Reaction of ROS with lipids leads to the highly damaging reaction, lipid peroxidation . Singlet oxygen reacts with unsaturated fatty acids and forms lipid hydroperoxides that break down to several products of lipid peroxidation . Lipid peroxidation induced by 102 has been implicated in the haemolysis of erythrocytes, damage to cardio­myocytes and degeneration of cellular membranes in different tissues. The other processes of biological

interest initiated by 102 include rancidity of oil s, spoilage of milk and coloured foodstuff exposed to light2,7 .8.

Lipid hydroperoxides (LOOH) are prominent non­radical intermediates of lipid peroxidation whose identification can often provide valuable mechanisti c information, e.g. whether a primary reaction is med i­ated by 102 or oxyradicals. Certain cholesterol­derived hydroperoxides (ChOOHs) have been used effectively in this regard, both in model systems and cells. Being more polar than parent lipids, LOOHs perturb membrane structure/function and can be dele­terious to cells. However, LOOHs can also participate in redox reactions, the nature and magnitude of which often determines whether the resulting peroxidati ve injury is enhanced or prevented. Enhancement may result from iron-catalyzed one-electron reduction of LOOHs, leading to free radical-mediated chain elon­gation, whereas prevention may reflect selenoperoxi­dase-catalyzed two-electron reduction of LOOHs to relatively non-toxic alcohols . An aspect of related research that is under intensive investigation is lipid peroxidationlLOOH-mediated stress signalling that may eventually lead to induction of antioxidant en­zymes and apoptotic cell death43

.

Participation of 102 in lipid peroxidation reactions can be established by analyzing the product of choles­terol oxidation. The hydroperoxides 3-beta-hydroxy-6-alpha-cholest-6-ene-5-hydroperoxide (5-alpha-00H), 3-beta-hydroxycholest-4-ene-6-alpha-hydroperoxide (6-alpha-OOH) and 3-beta-hydroxycholest-4-ene-6-beta­hydroperoxide (6-beta-00H) are derived specifically from 102 addition. During the photodynamic process, 5-alpha LOOH is photogenerated at a much greater initial rate and it also decays much more slowly dur­ing GSHlPHGPX treatment and hence more toxic to cells44

. The ratio of 7-LOOHl5-alpha LOOH or 7-LOOH/6-LOOH can be used as a highly sensitive in­dex of singlet oxygen vs free radical dominance in photodynamkally stressed cells45

. 5-Alpha LOOH has also been identified as a product in skin of rats pre­treated with oral doses of pheophorbide and subse­quent visible irradiation that have been known to in­duce photosensitive diseases in animals and humans. This shows the evidence for involvement of 10? in vivo in the etiology of disease46

. -

Various studies have also shown the involvement of 102 in lipid peroxidation induced by different pho­tosensitizers: that includes (1) In mitochondria of Sar­coma 180 ascites tumour exposed to the porphyrin derivative meso-tetrakis [4-carboxymethyleneoxy)

684 INDIAN J EXP BIOL, JUNE 2002

phenyl] porphyrin47.48 ; (2) melanotic M6 cell line ex­posed to bis(tri-n-hexylsiloxy)silicon phthalocya­nine49; (3) photodynamic treatment of pro myelocytic K562 cells in the presence of monoglucosy lporphyri n or hematoporphyrin50; (4) Fullerene C60 exposed to UV or visible light51 in rat li ver microsomes ; (5) mur­ine LI210 cells exposed to merocyanine 54052; (6) rat brain mitochondria exposed to 5,1O,l5,20-tetrak is[4-(carboxymethyleneoxy) phenyl] porphyri n53; and (7) liposomes exposed to hypocrellin A54 and other com­pOll nds55.56.

Lipid peroxidation can have both direct and indi­rect conseq uences . Normally cellul ar membranes are selectively permeable, hence allow on ly certain sol­utes to pass through. Th is ability is lost due to lipid peroxidation whose products modify the physical characteristics of biological membranes. Incorpora­tion of LOOH changes the physical structure of the membrane by decreasing the fluidity and increasing permeability. When free fatty acids get damaged membrane confirmation is lost and may lead to 'gaps' in the membrane. It can also cause cross-li nks be­tween two fatty acids , fatty acid and proteins etc. Thi s can eventually lead to change in membrane properties and loss of its bound enzymes. Lipid peroxidation can also resu lt in formation of several toxic byproducts that can attack other cellular targets, including DNA, away from the si te of generation. They can also alter cell signaling or act as 'toxic second messengers' that amplify damage. Such byproducts include 4-hydroxynonenal , malonaldehyde etc induce apoptosis. They form adduct with DNA and induce mutagenicity and carcinogenicity and induction of apoptosis57.

During lipid peroxidation the products formed such as LOOH can alter the physical characteristics of the membrane. Thus, the removal of the lipid peroxida­tion products from the membrane is necessary to re­pai r its damage and is accomplished by two separate enzymatic systems: the sequential action of phosphol­ipase A2 with glutathione peroxidase and phosphol­ipid hydroperoxide glutathione peroxidase. Phosphol­ipase A2 catalyzes the hydrolysis of the phospholipid hydroperoxides to the hydroperoxy fatty acids. Once released, the fatty acid hydroperoxides may undergo a reaction with glutathione peroxidase to form stable, reduced hydroxy products. A second enzymatic sys­tem eliminates phospholipid hydroperoxides from lipid membranes through the direct reaction of phos­pholipid hydroperoxide glutathione peroxidase with the esterified phospholipid hydroperoxides58.

Proteins Reaction of 10 2 with proteins IS more selective

yieldi ng specific products . Among the amino ac ids histidine, tryptophan, meth ionine and tyrosi ne are more reactive towards this ROS . Oxidation of these ami no acids results in su lphoxides and short-lived endoperoxides that may be toxic to other cells. If ROS is generated in solution , it may lead to non-specific (g lobal) protein damage, whi le if it is 'site-specifi­cally generated' can lead to site-specific or localized damage. Global damage can be measured by estimating protein carbony ls. In the loca li zed damage, the defensive action of scavengers to remove ROS decreases dramatically, since they are unable to access the microenvironment. The study of oxidation of proteins has gained momentum in recent years and has been linked to various diseased states and the process of ageing. Lipofuscin, an aggregate of peroxi­dized lipid and proteins, accumulates in Iysosomes of aged cells, brain cells of patients with Alzheimer's disease and in iron-overloaded hepatocytes. The carbonyl content of protein in rat hepatocytes increases with age. Oxidative inacti vation of several enzy mes has been associated with age ing and in pathological states like ischemia-reperfusion . Singlet oxygen can inactivate proteins as exemplified by enzymes of citric ac id cycle exposed to intracellul arly generated 10 2 and crystallin proteins of the eye exposed to sunlight. Such oxidation of crystallins leads to formation of high molecular weight cross­links that may eventually result in catarad ·2.8.59.60.

DNA Reaction of 102 with DNA can lead to strand

breaks and formation of altered bases. 102 was gene­rated by 3 different methods namely i) microwave discharge, ii) photosensitization with rose bengal im­mobilized on a glass plate, and iii) chemical genera­tion using the thermal decomposition of the endoper­oxide of naphthalene dipropionate3.57.61.62. Exposure of single-stranded bacteriophage M13 DNA to 102 led to a decrease in transforming activity. Loss of such activity was doubled following replacement of H20 in the buffer by D20, increasing the life time and conse­quently, the diffusion path-length of the 102 gener­ated. Single-stranded DNA was more susceptible than double-stranded DNA. Later studies have shown that reaction of 102 with bases like guanosine, the most susceptible base, produced through cycloaddition mechanism showed a 7-fold higher reactiv ity with single-stranded as compared to duplex 8-hydroxyde-

DEVASAGA YAM & KAMAT: SINGLET OXYGEN AND ITS BIOLOGICAL EFFECTS 685

oxyguanosine63. Recent resu lts demonstrated that 102, when released within cells, is able to oxidize cellular DNA directly64.

Devasagayam et al. 65·68 have used several simple biological systems for studying the effect of photo­sensitization;J02 and its possible prevention by natu­ral and/or dietary compounds that function as antioxi­dants . One of the model systems used for studying the mechanisms and modulation of DNA damage caused by photosensitization is plasmid DNA. After exposure in presence and absence of different modifiers the DNA was subjected to agarose gel electrophoresis. Gel is photographed by a polaroid camera and the resulting photo-negative is scanned in a scanning den­sitometer. In control DNA most of DNA gets sepa­rated as Form I (supercoi led form) of DNA. When the DNA gets exposed to photosensitization , for example in presence of methylene blue plus visible light, there is a significant 'increase in the relative amount of Form II that results from single-strand breaks. Dam­aging effect of 102, generated by thermal decomposi­tion of naphthylidine dipropionate on DNA and its protection by several natural antioxidants has also been studied65.68 .

Studies showing the ab ility to induce strand breaks was performed wi th plasmid pBR32266,69,70 , For the formation of single-strand formation, a second-order mechanism was suggested, as the rate of single-strand breaks is proportional to the square of the 102 produc­tion . Studies usi ng the combination of scavengers of ROS and D20 have conclusively proved that 102 was indeed the species responsible for strand break forma­tion during the thermal decomposition of the endoperoxide of naphthalene dipropionate7,67 .

Singlet oxygen induced strand-breaks occur spe­cifically at guanosine residues and there was no selec­tivity among these bases. Such strand-break formation was also accompanied by formation of 8-hydroxy­deoxyguanosine68. These reactive species react with guanine moiety in nucleosides and DNA. The oxida­tion products include 8-hydroxydeoxyguanosine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy­Gua). Presence of 8-hydroxydeoxyguanosine in DNA can have serious biological consequences. One such event is random termination of DNA replication oc­curring at the position of the modified guanine residue and at its neighbouring bases resulting in misreading by DNA polymerase71 . These errors in DNA replica­tion can eventually result in mutagenesis and carcino­genesis. Singlet oxygen can also causes alkali-labile sites and single-strand breaks in DNA. The biological

consequences associated with 102 induced DNA dam­age include loss of transforming ability in plasmids and bacteriophages, mutagenicity and genotoxici ty72.

The favoured oxidation of guanine within DNA may be explained by its lower oxidation potential with respect to that of other bases73

. DNA strand breaks induced by 102 seem to be initiated by its reac­tion with guanine which is hydroxylated at the 8-position, leading to endoperoxide formation 66. Re-

74 1 cently, Chanon et al. have proposed that O2 react-ing with guanosine or deoxyguanosine part of nucleo­tides does not, by itself, cause DNA cleavage. The strand break originates at the endoperoxide stage whenever this link evolves into an O-centered radical. This radical is then in a good spatial position to ab­stract an hydrogen intramolecularly from the ribose or deoxyribose part of the nucleotide. The carbon­centered radical thus formed on the sugar part may lead to strand break either by a p-scission mcchanism or by a homolytically induced lysis.

Singlet oxygen has been shown to be the mediator of DNA damage induced by UVA (320-400 nm) and induce the format ion of 8-hydroxydeoxyguanosine75 . Similar participation of 102 was shown during reac­tion of peroxynitrous acid and hydrogen peroxide76. Such role for 102 also has been assigned during pho­tosensitization induced by photosensitizers such as methylene blue77-80, rose bengal81 meso-tetrakis [4-carboxymethyleneoxy) phenyl] porphyrin82 meso­tetrakis [3-carboxymethyleneoxy) phenyl] porphyrin ; meso- tetrakis [3,4-bis(carboxymethyleneoxy) phenyl] porphyrin83, meso-tetra(4-N-methylpyridyl)porphyrin84

,

meso-tetra( 4-sulphonaoEhenyl)porphyrin57; cationic tetrauthenated porphyrin 5 and the fullerene C6Q (ref. 86).

There are some natural barriers designed to protect the genome from radical attack. These include com­partmentalization of the sensitive target molecules and shielding of nonreplicating DNA by histones and polyamines . If protection of DNA is not successful , cellular regulatory mechanisms such as induction of apoptosis and inhibition of cell cycle progression may prevent transfer of damaged DNA to the offspring. Alternatively, DNA repair processes can correct the damage. Excision repair is performed by enzymes such as DNA glycosylases and AP endonucleases that occur before replication, while postreplication repair provides a method for repairing lesions during or after replication87.

Major repair occurs by excision of the oxidized de­oxyguanosine moieties by Fpg protein (formami­dopyrimidine-DNA glycosylase), preventing mismatch

686 INDIAN J EXP BIOL, JUNE 2002

of 8-hydroxydeoxyguanosine with dA, which would generate G:C to T:A transversions43

.58. More recent studies have showr. that the repair of 102 -i nduced DNA lcsions requires several enzymes of the nucleo­tide and base excision repair pathways, including ex­onuclease III and endonuclease IV that are known apurinic/apyrimidinic endonucleases in Escherichia coli. The other types of mutation induced by 102 can be G:C to C:G transversions. Exonuclease In may act on the repair of 102-induced lesions altering the DNA repair sequence specificit/8

. Biological protection against such damage in the form of natural antioxi­dants is afforded by compounds like lipoate, carote­noids, flavonoids, curcumin, tocopherols and the food-flavouring agent vanillin2.67.68.80.89.

Cellular defenses against 102 and damage induced The defenses to counteract the potentially hazard­

ous reactions initiated by 102 include all levels of pro­tection namely, prevention, interception and repair. Prevention mainly deals with the alteration of reac­tions that result in 102 generation involving both en­zymatic and non-enzymatic reactions. This can occur in various ways such as reduction and availability in the amount/availability of endogenous sensitizers and/or substrates for photosensitizing/enzymatic reac­tions that generate such reactive species. Apart from this and the repair enzymes that can take care of the 102-induced lipid, protein and DNA damage men­tioned earlier, tissues also contain other lines of de­fenses in the form of antioxidants capable of quench­. 10 90 mg 2.

There are two types of quenchers based on the mechanism of action: 1) compounds such as carote­noids and nickel complexes quench 102 by energy transfer with high rate constants, generally in the re­gion of 109 - 1010 M·1s·1; 2) compounds like DABCO (diazabicyclo(2.2.2)octane), phenols, sulphides and azides are known to quench 102 by electron transfer (or charge transfer) mechanisms with lower rate con­stants, generally in the region of 106_108 M·1s·1. Al­most all quenchers of 102 are compounds with low oxidation potential and will certainly react with other strong oxidants in the system. Hence there may not be "specific 102 quenchers" to 'characterize' the in­volvement of 102 reactions, especially in biological

41 systems -.

There is an increasing interest in the role of diet nu­trition in pathogenesis and possible prevention of can­cer. The question has been raised whether ~-carotene

may have anti carcinogenic properties independent of its provitamin activity. An inverse relationship be­tween ~-carotene intake and the incidence of certain types of cancer has been observed. Animal experi ­ments have revealed anticarcinogenic properties of carotenoids 8.91. The anticancer ability of carotenoids have been attributed to physical quenching capacity of 10 2 was first described by Foote and Denny92. How­ever, oxidation products of ~-carotene can behave as prooxidants under certain conditions and promote

. . 93 carcmogenesls . Potential cellular and plasma antioxidants that pro­

tect against 102 include carotenoids, tocopherols, thiols and small molecular compounds such as car­nosine, bilirubin etc. Among the biological com­pounds (Table 3), carotenoids are rhe most efficient quenchers. Though the quenching abi lities of the tested carotenoids are close to the limit of diffusion control, there are considerable differences. Lycopene (present in tomato), the biologically occurring open­chain isomer of ~-carotene, shows the greatest quenching ability. Capsorubin, present in chillies show a very high amount of quenching94

. The other

Table 3-Singlet oxygen quenching ability of various antioxidants

Compound Rate constant - 106M· l s·1

Lycopene 9,000 y-Carotene 7,300 Canthaxanthin 6,100

~-Carotene 4,100

Methylbixin 3,000 Lutein 2,300 Norbixin 2,300 Bixin 1,800 Bilirubin 1,500 Uric acid 360 Azide 200 Nicotinamide 180 Ascorbic ac id 160 Ubiquinone 158

~ Tocopherol 153

y·Tocopherol 138

(X- Tocopherol 130 Lipoale 60 Vanillin 60 0-Tocopherol 53 Imidazole 40 Hi stidine 3 1 Caffeine 29 Hi stamine 28 Methionine 9 Cysteine 8 Thiourea 4 Glutathione 2

DEVASAGA YAM & KAMAT: SINGLET OXYGEN AND ITS BIOLOG ICAL EFFECTS 687

biologically occurring carotenoids like y-carotene, a ­carotene, astaxanthin, zeaxanthin, lutein and crypto­xanthin also showed a high extent of quenching8,42.95.96. The quenching by carotenoids depends largely on physical quenching and to a lesser extent by chemical reaction.

Tocopherols are less efficient than carotenoids, whereas thiols and related sulphur-compounds are the least effecti ve. The concentration of latter group of compounds in tissues however, is several fold higher than carotenoids. Baltschun et al.97 have determined the bimolecular rate constants of 27 natural and novel synthetic carotenoids. Among these compounds, an empirical correlation between nn* excitation energy and the structure of the carotenoid was found. The quenching abilities depend on the excitation energy of their transition at long wavelengths in a characteri stic way showing as limiting factors either the thermal Arrhenius activation or the diffusion-controlled rate.

Carotenoids are abundant in many fruits and vege­tables and they play diverse roles in photobiology, photochemistry and medicine. They react with '02 as well as other ROS of biological significance. They can also interact with other antioxidants and under certain conditions behave as prooxidants98. The ra­diomodifier buthionine sulfoximine quenches '02 with a rate constant of d '/~ 107 M" s'l. (ref.99). There are also several other compounds that quench '02. These include probucol (l06M,'S' '), phenolic, nitroge­nous and sulphur-containing compounds'oo, squaiene'OI,'02 and lipoic acid, 03, ,o5.

Several cellular antioxidants are capable of pre­venting damage caused by '02 to biological mole­cules. Carotenoids and tocopherols are able to pre­vent/delay the peroxidation of lipids induced by '02 in cellular membranes. Several natural antioxidants play key roles in the preservation of membrane integrity. Roles of vitamins A, C and E in this aspect are well documented. Very little information is known about the function of Vit B3, nicotinamide against oxidative damage. Kamat and Devasagayam '04 demonstrated for the first time that nicotinamide, an endobiotic nicotinamide exhibits excellent antioxidant ability against photosensitization-induced '02 with the rate constant of 1.8 x 108 M-'s-'.

They devised a simple set-up for exposing biologi­cal samples to photosensitization. The tissue sample is kept in a 'trap' maintained at 37°C. Oxygen is bub­bled through and a visible light source is placed at about IS cm from the trap. Using this the component

requirement fo r lipid perox idation whose product was estimated as thiobarbituric acid reacti ve substances (TB ARS) was studied. Lipid perox idation in absence of O2, light or methylene blue was negligible. Whereas when all the th ree components are present, the peroxidation is very high. Another poin t to be noted is if O2 + light is given there was considerable peroxidation. This indicates that there are certain en­dogenous compounds present in microsomes that can act as sensitizer '04. In these studies they have exam­ined the antioxidant ability of certain natural and die­tary components as protectors of membranes against ox idati ve damage induced by '02. Among the com­pounds examined chlorophyllin, caffeine, nicotina­mide, vanillin and tocotrienols from palm oil were found to be effective. Chlorophyll in, the sodiu m­copper salt and water-soluble analogue of the ubiqu i­tous plant pigment chlorophyll has been attributed to have several benefici al properties. It is highly effec­tive in protecting rat liver mitochondria against photo­sensitization even at low concentrations. It also has a fairly high rate constant with '02 in the order of l.3 x 108M" s" (ref. lOS). Similarly caffeine present in coffee, tea and cola-based soft-drinks also protects rat liver microsomal membranes against '02 and also re­acts with this reactive species (rate constant of 7.3 x 107 M-'s-'). Vanillin, the commonly used food flavouring agent also protected subcellular mem­branes, in the form of rat liver mitochondria against damage induced by photosensitization (rate constant 6x 107 M-'s-'). Nicotinamide (vitamin B3) is an effec­tive protector of both rat liver microsomes and rat brain mitochondria against photosensitization '04, '06. In addition to this property nicotinamide also has other beneficial effects such as like chemoprevention and induction of drug metabolizing enzymes'07,I08. Toco­trienols from palm oil that are vitamin E derivatives also show membrane protective properties in rat brain microsomes and rat liver microsomes' 09'"'. Biological antioxidants such as lipoate, methionine, flavonoids, related polyphenols, /3-carotene, a-tocopherol and curcumin from turmeric also prevent DNA damage induced by '02.

There are several biologically useful effects and potential applications of '02 (Table 4) ,

Beneficial effects of l02-the photodynamic therapy If '02 can be selectively generated in diseased ti s­

sue, such as tumor, it can behave as a 'therapeutic agent', in controlling the disease. Photodynamic ther­apy (PDT) is the combination of light and light sensi-

688 INDIAN J EXP SIOL, JUNE 2002

Table 4- Potential appl ications of singlet oxygen

I Defense mechani sms : phagocy tosi s and degradation of endogenous hallucinogens 2 Hormonal ac tivity of prostaglandins 3 Photodynamic therapy of cancer, atherosclerosis, skin diseases etc. 4 Inactivat ion of pathogens like viruses, bacteria etc. in blood 5 As a disinfectant 6 Invol ved in cell signaling 7 As a bleaching agen t 8 For water treatment 9 For pest control using photoactive pesticides

10 For synthetic reactions II In PUV A therapy for psoriasis 12 Chemiluminesccnce of spec ific biocompounds

tive agents (such as porphyrins) in an oxygen-rich environment. Porphyrins, a component of hemoglobin can absorb energy from photons and transfer this en­ergy to surrounding oxygen molecules. Toxic oxygen species such as si nglet oxygen and free radicals are thus formed. These species are very reactive and can damage proteins, lipids, nucleic acids and other cellu­lar components. Porphyrins utilize energy from light

d · . 11211 3 to pro uce tOXIC oxygen speCIes . . Modern PDT originated at the turn of the century

in Germany. Researchers experimenting with self­injection of porphyrins noted sunburns due to photo­dynamic reactions in their skin. Derived from animal hemoglobin, two forms of porphyrin are well known: hematoporphyrin derivative (HPD) and porfi mer sodium, (Photofrin), which is in Phase III clinical tri ­als has received approval in Canada for use with bladder carcinoma where treatment with BCG vaccine has failed . Photofrin has been approved in other coun­tries for treatment of esophageal cancer and lung can­cer. These first generation photosensitizers display prolonged and generalized photosensitivity of the skin as their primary side effect. Second generation photo­sensitizers exhibit far less photosensitization and are now in early clinical tri als (one example is BPD veI1eporfin). Which was recently in Phase IIII clinical tri als for primary skin carcinoma, cutaneous lesions where cancer has metastasi zed to the skin, and chronic stable plaque psoriasis.

Lasers are the primary light source for activation of porphyrins because laser light is monochromatic (ex­act ly one colour), coherent (light waves are parallel permitting precise focusing), and intense (allowing for shorter treatment times). Light Emitting Diodes (LEOs) and florescent light sources are now being used as alternative light sources as they are more con­venient than lasers but do result in longer treatment times.

A typical PDT session involves (i) intravenous in­jection (i.v.) or topical application of a photosynthe­sizing agent such as a porphyrin; (ii) permit time for systemic porphyrins (i. v. injection) to be cleared from normal tissues and be preferentially retained by rap­idly growing tissues (e.g., cancer or psoriasis), or for topical porphyrins to be absorbed by the skin; (iii) application of light to provide the catalyst for chemi­cal reactions; (iv) generation of toxic oxygen species in illuminated tissues and (v) tissue damage usually resulting from damage to vasculature giving rise to regression of diseased tissue like cancer. Photody­namic therapy (PDT) is not only just an application of technology, but may be considered as a completely new concept. A variety of different types of tumors also respond to PDTI1 2. 11 3. Photodynamic effect has also been used in sterilizing blood and blood products, as antiviral agents and in the control of insect pests 91. Furocumarin derivatives (psoralens) were used as photosensitizers during UV treatment of psoriasis and other types of skin diseases. This reactive species generated in these studies, may be a key component in fe . h 114-I?O e lectlllg t e treatment -.

The mechanisms involved in cell killing in PDT may be related to damage to mitochondria and induc­tion of apoptos is. It is believed that the generation of singlet oxygen helps in inducing cell death. At higher concentrations singlet oxygen and other ROS may induce death by necrosis, while at lower doses apop­tosis predominates. The former is the result of over­whelming incident stress, while the latter involves the cell's systemic self-destruction without affect ing the

d· . P I surroun lIlg tIssue - .

Signalling effects of singlet oxygen Recent studies showed that 102 is involved in cell

signali ng l22. This species generated during PDT or exposure to UV A, was shown to induce a series of

DEV ASAGA Y AM & KAMAT: SINGLET OXYGEN AND ITS BIOLOGICAL EFFECTS 689

genes involved in signaling cascade such as JNK and p38 MAP kinase and NF-KB. Both may contribute to induction of enzymes involved in signaling pathways. Three pathways have been shown to be induced by '02, the AP-l, NF-KB, and AP-2 pathway. The cell membrane appears to playa role as one of the primary targets for '02.

Conclusion Though the biological relevance of '02 has been

realized only recently a large number of studies show that '02 is being generated by a number of biologi­cally important reactions with significant implications in disease development. Damage caused by '02 can be prevented by many natural antioxidants. This spe­cies also has several potential applications including in cancer therapy. At low levels '02 can be a signaling molecule. Newer approaches to the study of '02 can yield rich dividends.

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