genotoxicity of active oxygen species in mammalian cells

Mutation Research, 195 (1988) 215-230 215 Elsevier

MTR 07244

Genotoxicity of active oxygen species in mammalian cells

R o g e r i o M e n e g h i n i

Department of Biochemistry, Institute of Chemistry, Unioersity of SSo Paulo, SSo Paulo, CP 20780 (Brasil)

(Received 28 April 1987) (Revision received 11 August 1987)

(Accepted 25 August 1987)

Keywords: Active oxygen species; Mammalian cells; Genotoxicity; Oxygen molecule, ground state

Because the oxygen molecule has in the ground state 2 unpaired electrons with the same spin number in 2 external orbitals, it can hardly accept a pair of electrons from the donor, since these usually have opposite spins. This is why the oxidation of organic material, although thermodynami- cally spontaneous, goes very slowly at ordinary conditions, requiring an initial supply of energy to produce radical species by homolytic excisions which then react promptly with oxygen.

One-electron transfer is a way of speeding up reactions of ground state oxygen and this is the reason why it reacts fast with radical species (having one or more unpaired electrons in external orbitals) or with transition metals (having upaired electrons in internal orbitals). Dioxygen is itself a radical species since it has 2 unpaired electrons with the same spin number (i.e. triplet state). Upon supply of energy and under specific conditions triplet oxygen can be transformed into singlet oxygen, in which the two electrons have opposite spins. Singlet oxygen is more reactive than triplet oxygen but its formation in biological systems seems to be restricted to very specific conditions (for instance, illumination of some pigments, like chlorophylls and porphyrins, in the presence of oxygen). Other active oxygen species, generated by monoelectronic reduction of dioxygen seem to

Correspondence: Dr. Rogerio Meneghini, Department of Bio- chemistry, Institute of Chemistry, University of $5o Paulo, S~o Paulo, CP 20780 (Brasil).

be more conspicuous in biological systems. These species are superoxide radical anion (02) , hydrogen peroxide (H202) and hydroxyl radical (OH'):

- - ° - ; o w 02 ~ 0 2 ~ H2021_~OH_.

I (I)

In general, aerobic cells are capable of reducing 0 2 t o water without the formation of intermediates. The enzyme responsible for this process is the copper-containing cytochrome oxidase, whose mechanism of action is still poorly understood. However a small fraction of cellular dioxygen is reduced by monovalent steps, with the concomitant production of the intermediate active oxygen species (AOS). The OH radical is specially reactive, attacking virtually any organic compound with kinetics which are close to diffusion- limited processes. The reason why monoelectronic reduction occurs in the cell is not well understood. Some recent investigations have shown that microsomal metabolism of certain compounds, like al- cohol, may require OH radical as oxidant (Klein et al., 1983). The possibility of a physiological role for the reactive intermediates of oxygen reduction is well exemplified by the controlled production of O 2 by the NADPH oxidase bound to membranes of phagocytic cells; this enzyme is known to play an important role in the killing of invading micro- organisms (Michell, 1984). Several enzymes are

0165-1110/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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known to reduce dioxygen into 0 2 and /o r H202 (Halliwell and Gutteridge, 1984). However, the accumulation of these species in the cell would certainly be deleterious and has to be prevented. The cells are equipped with enzymatic and non- enzymatic mechanisms for this task. Superoxide dismutases (SOD), are enzymes widely distributed in aerobic organisms, which accelerate the dismutation of 0 2 (Fridovich, 1978):

2 H + + 2 O f superoxide dismutase ) H202 + 02 (II)

Hydrogen peroxide itself is not risk free; its decomposition is catalyzed by catalase and its reduction by glutathione peroxidase:

2 H202 catalase> 2 H20 + 02 (III)

H202 + 2 RH glutathione peroxidase ) R - R + 2 H20 (IV)

Since reactions of OH radical with organic compounds exhibit kinetics close to diffusion- limited processes it is not feasible to scavenge this species by means of an enzymatically catalyzed reaction. Antioxidant compounds may, however, provide such scavenging action due to the high rate constant of their reactions with OH radical. The simultaneous action of antioxidant enzymes and antioxidant small molecular weight compounds (like glutathione, ascorbic acid and vitamin E) constitutes the first line of defence against the reactive oxygen species, helping to maintain their steady-state concentrations at levels that are compatible with cellular integrity.

Increased cellular production of reactive oxygen species has been noticed under different stressing conditions, such as upon exposure to visible and ultraviolet light (Hoffmann and Meneghini, 1979a; Parshad et al., 1978), ionizing radiation (Hart, 1972), during metabolism of certain compounds such as aromatic hydrocarbons (Lorentzen and Ts'o, 1977), quinones (Nohl et al., 1986) and paraquat (Hassan and Fridovich, 1977).

Under such circumstances lesions may be produced in important cellular components. Obvi- ously, the defence provided by cellular antioxidants may be overwhelmed and then undesirable reactions between oxygen species and cellular

structures take place. There is an impressive list of degenerative processes that may have etiologic origin in events mediated by oxygen species. It comprises Parkinson's disease (Cohen and Heik- kila, 1974), ischemic damage (McCord, 1985), emphysema (Dooley and Pryor, 1982), arthritis (Mc- Cord, 1974), mutagenesis (Moody and Hasan, 1982), cancer (Ames, 1983; Cerutti, 1985; Borek and Troll, 1983) and senescence (Cutler, 1984). It has been considered by many investigators that even at normal steady-state concentration oxygen species may be continuously producing cumulative damage in specific targets which may eventually lead to tissue degeneration.

From the point of view of DNA as a target, it is important to consider mutagenesis, cancer and senescence. Mutagenesis has by definition DNA as the relevant target. It is now unquestionable that D N A is a structure whose alteration may lead to the initiation and promotion of cancer. Free radicals generated by metabolic processes may damage gene-regulatory sites and cause cellular inefficiency which may ultimately lead to death (Cutler, 1984). Not surprisingly many investigators have focused their interests on the mechanisms of production of DNA damage by reactive oxygen species. I shall attempt critically to review recent results concerning (i) chemical processes that lead to the production of oxygen species capable of attacking DNA, (ii) DNA lesions produced in this way and their repair, and (iii) biological endpoints arising from these processes. Most of the results to be discussed come from experiments carried out with mammalian cells in culture.

Cellular generation of oxygen free radicals with relevance to DNA

Excellent review articles have been published recently with respect to the mechanisms of oxygen radical production in cellular systems. The reader is referred to the articles of Halliwell and Gut- teridge (1986) on oxygen radicals and transition metals, Fridovich (1986) on the protection afforded by superoxide dismutase, Ames (1983) on the production of oxygen radicals by dietary compounds and Slater (1985) on the production of oxygen radicals in tissue injury. I shall only dis-

cuss briefly those aspects relevant to DNA. When DNA is exposed to a hydrogen peroxide solution no modification of molecular weight occurs (Hoff- mann and Meneghini, 1979b). If instead DNA is exposed to a source of 0 2 strand breaks are observed. However, catalase fully protects DNA from the O2- action (Lesko et al., 1980; Brawn and Fridovich, 1981; Mello-Filho and Meneghini, 1985). These results can be explained if 0 2 and H202, produced from O~- by spontaneous dismutation, are simultaneously required for strand break production. The chemical process which better expresses these requisites is the Haber - Weiss reaction (Haber and Weiss, 1934).

H202 + O~- ~ O H ' + O H - + 02 (V)

This reaction produces an OH radical which is the species that ultimately attacks DNA. In fact, if DNA is exposed to a source of O~- in the presence of an OH radical scavenger no strand breaks are observed (Lesko et al., 1980; Brawn and Frido- vich, 1981; Mello-Filho and Meneghini, 1985). These scavengers have structures that render them capable of reacting rapidly and in a relatively specific manner with the OH radical and therefore they may provide protection to other targets. Ethanol, mannitol, thiourea, benzoate, and dimethyl sulfoxide, are OH radical scavengers frequently used.

As a matter of fact, reaction V is thermody- namically feasible but kinetically very slow unless certain transition metals are present (Halliwell, 1978; McCord and Day, 1978). Iron is an excellent catalyst and through its mediation reaction V can be understood as the sum of two other reactions:

0 2 + Fe3 + ~ Fe2 + + 0 2 (vi)

Fe 2+ + H202 ~ O H ' + O H - + Fe 3+ (VII)

0 2 + H202 -+ 0 2 -[- O H - + OH" (v)

Reaction VII has been known for a long time and is referred to as the Fenton reaction (Fenton, 1893). Reaction VI shows that O 2 plays its role in the production of DNA damage as an iron re-

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ductant. In fact, the concept has gained acceptance that O 2 is not a good oxidant in aqueous solution at pH 7.0, although when embedded in membrane it is a nucleophile and will oxidize phospholipids (Halliwell and Gutteridge, 1985).

Iron is the most abundant transition metal in biological systems, but in general it is complexed with proteins, as in hemoglobin, transferrin, ferritin and cytochromes. Transferrin (Halliwell and Gutteridge, 1986) and hemoglobin (Sadrzadeh et al., 1984) are poor Fenton reactants, whereas ferritin seems to produce OH radical from H202 (Bannister, 1984). Apparently, chelates in which a coordination site on iron remains open or loosely associated with water are catalysts of the Fenton reaction, like free iron, EDTA-Fe , ATP-Fe , bleomycin-Fe, whereas hindrance of all coordination sites effectively blocks OH radical production (Graf et al., 1985). To the latter category pertains DETAPAC (diethylenetriamine pentaacetic acid) and desferal; o-phenanthroline and a,a '-bipyridine form iron chelates in which Fe II is strongly stabilized and cannot be oxidized by H202 (Mello-Filho and Meneghini, 1985).

At least 2 other biological transition metals, copper and cobalt, are Fenton reactants too (Samuni et al., 1983; Moorhouse et al., 1985). Most of the biological copper is bound to proteins, like ceruloplasmin and albumin, or to amino adds, in which forms it may participate in the Fenton reaction and the resultant OH radical may attack the ligand to which the copper is bound (Csapski, 1984).

It is important to point out that although the Haber-Weiss reaction has been shown to explain the production of DNA damage by reactive oxygen species in vitro, to prove this in vivo is far more difficult. In addition, we must not leave the impression that the Haber-Weiss reaction is the only possible relevant process to produce oxygen radicals capable of damaging DNA. In this context (i) ascorbic acid has been shown to be an important reductant that can substitute O f in reaction VI (Samuni et al., 1983; Winterbourn, 1979; our unpublished results); (ii) alkoxy radicalS can be formed instead of the OH radical, when iron reacts with alkyl peroxides (Halliwell and Gutteridge, 1985); alkoxy radicals may react with DNA to produce lesions; (iii) semiquinones can

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also reduce transition metals and have been shown to react with H202 to produce OH radical in an "organic Fenton reaction" (Nohl et al., 1986):

HR-~O'+ 1-1202 ~ R'~-O + OH'+ H20 (VIII)

Both semiquinones and H202 are normal metabolic products and may thus be a continuous source of OH radical production in the Cell. The need for a metal catalyst in this reaction is still an open question; (iv) suggestions of other species instead of an OH radical being formed from the Fenton reactants have been put forward. These include ferryl (FeOH 3+) and perferryl (FeO ÷) radicals (Walling, 1982; Halliwell et al., 1982). The latter has a much lower oxidizing capacity than the OH radical, whereas the former, if a product of a Fenton reaction, should attack spin traps to produce the proper radical signal in EPR and this has not been demonstrated (Halliwell and Gutteridge, 1985). Therefore it is now generally accepted that the OH radical is the oxidizing species produced in Fenton reaction.

Structural alterations produced in DNA by reactive oxygen species

It has been known for a long time that reactive oxygen species produce DNA damage (Rhaese and Freeze, 1968). Apparently 0 2 and H202 alone cannot oxidize D N A (Lesko et al., 1980; Brawn and Fridovich, 1981; MeUo-Filho and Meneghini, 1985). The hydroxyl radical is a powerful DNA oxidant but unfortunately the chemistry of this reaction is relatively unexplored, and the results from one type of experiment cannot be gener- alized because they depend on the specific form by which the OH radical is generated. Thus, although it has been shown that DNA damage produced by ionizing radiation is to a great extent mediated by the OH radical produced during water radiolysis (Repine et al., 1981a) the DNA structural alterations produced during this reaction cannot be necessarily extrapolated to situations in which the OH radical is generated by other mechanisms. This is because, in addition to the OH radical, several other species are formed by water radiolysis, like the solvated electron and the hy-

drogen atom, which may themselves produce DNA damage. In experiments in which DNA was exposed to H202 plus iron (Rhaese and Freese, 1968) or to plain H202 (Demple and Linn, 1982) - - in this case traces of iron were certainly present to elicit a Fenton reaction - - the major products observed were thymine glycol, single-strand breaks and base-free sites, predominantly apurinic sites. In DNA exposed to tumor promoter-activated polymorphonuclear leukocytes, which constitutes a source of oxygen radical, 5 hydroxymethyl-2'- deoxyuridine is formed in a 1:2 ratio to thymi- dine glycol (Frenkel et al., 1986). When the OH radical is generated by a Fenton reaction the type of DNA lesion may be considered to depend on the topological localization of the metal. One can imagine two extreme situations, one in which the metal is freely soluble, floating around the DNA molecule. This situation has been studied by exposing DNA to EDTA-Fe complex plus H202 and the results have shown that the OH radical breaks the backbone of DNA with almost no sequence dependence (Tullius and Dambroski, 1986). Very probably the main targets are the deoxyribose sugars arrayed along the surface of DNA. Secondary reactions of the resulting deoxyribose-centered radicals eventually cause the backbone to break, in essence by disintegration of the sugars themselves (Hertzberg and Dervan, 1984). In another situation the metal may be tightly bound to DNA (Shires, 1982). If this metal complex is a Fenton reactant the OH radical would be generated very near to the site of reaction in DNA, and perhaps due to the geometry of the complex, specific sites of the DNA molecule could constitute favorable points of attack for the OH radical. A circumstance in which the catalyst metal of the Fenton reaction is bound to the OH radical target has been referred to as a site-specific Fenton reaction (Samuni et al., 1983). There are suggestions that it occurs in the radiation-induced inactivation of T7 phage in the presence of Cu 2÷ (Samuni et al., 1983) and in the formation of DNA strand breaks when human fibroblast nuclei are exposed to H202 (Meneghini and Hoffmann, 1980). A characteristic of a site-specific Fenton reaction is that its effect is suppressible to a lesser extent by OH radical scavengers because the sites of origin and of reaction of the OH radical are too

close together (Samuni et al., 1983). As a corollary, lack of effect of the OH radical scavenger cannot be necessarily taken as an indication that a Fen- ton reaction is not involved in the process.

Certain natural or synthetic compounds can bind simultaneously to metals and DNA. In some of these ternary complexes iron or copper can undergo redox cycle very efficiently giving rise to oxygen species that attack DNA in site (Dervan, 1986). The complex Cu +-o-phenanthroline-DNA has such characteristics (Que et al., 1980). Other metals do not substitute for copper and the ultimate DNA-damaging species appears to be the OH radical (Que et al., 1980). DNA degradation seems to proceed via oxidation of the deoxyribose and primary sequence specificity is apparent in the scission reaction (Pope et al., 1982). Interest- ingly, Z-DNA is completely resistant to Cu+-o - phenanthroline (Pope and Sigman, 1984). The an- tibiotic adriamycin forms a ternary complex with Fe 3÷ and DNA with a strong catalytic capacity for reducing O 2 by thiol groups, leading to generation of 02 , H202 and OH radical. This seems to be responsible for DNA cleavage which occurs non-randomly with the generation of defined 230 base pair fragments (Eliot et al., 1984). Bleomycin is an antineoplastic drug which also forms a ternary complex with iron and DNA, promoting its cleavage (Burger et al., 1982). There have been claims that the OH radical does not mediate this cleavage on the basis of a lack of protection by OH radical scavenger (Rodrigues and Hecht, 1982). But again, this may be a typical example of a site-specific Fenton-like reaction in which acces- sibility of scavengers to OH radical is very difficult. It is interesting that several classes of proteins involved in nucleic acid binding contain sequences that potentially could form metal-binding domains. These metal-binding units might func- tion so that relatively short sequences can form independently structured domains, helping them to form stable DNA-protein complexes (Berg, 1986). Copper and iron are ions that could potentially bind to these proteins.

As opposed to purified DNA, exposure of cells to H202 leads to DNA strand breaks. When cells are exposed to H202 or to a mixture of H202 plus 02, provided by the xanthine oxidase-catalyzed oxidation of aldehydes, DNA strand breaks are

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formed. In both cases, catalase affords full protection (Hoffmann and Meneghini, 1979b; Mello- Filho and Meneghini, 1984, 1985; Meneghini and Hoffmann, 1980; Ward et al., 1985; Birboim and Kanabus-Kaminska, 1985; Schraufstatter et al., 1983; Bradley and Erickson, 1981; Wang et al., 1980; Hoffmann et al., 1984), but in the case of strand breaks produced by the xanthine oxidase system no protection is provided by superoxide dismutase (Mello-Filho and Meneghini, 1984). Therefore, externally generated 0 2 plays no role in DNA damage production other than to dis- mutate into H202. This latter species, as opposed to Of , diffuses freely into the cell where it is destroyed very efficiently by catalase and glutathione peroxidase. In fact 5 × 105 V79 Chinese hamster fibroblasts destroy in 45 min 50% of the hydrogen peroxide contained in 5 ml of medium at a concentration of 10 -4 M. This same rate of H202 consumption is observed for 3T3 mouse and VA13 human fibroblasts. Nevertheless, some of the H202 reaches the nucleus and cleaves DNA. The extent of strand break production by H202 is dependent on the cell type and seems to be very species-specific. A 30-rain treatment of 5 × 105 cells at 37°C with 2 ml of 2 x 10 -4 M H202 produced 12, 4 and 2 single-strand breaks/108 dalton of DNA, respectively, for human, mouse and Chinese hamster fibroblasts (Hoffmann et al., 1984).

Protection by the OH scavenger dimethyl sulfoxide is only observed at concentrations in the molar range (Ward et al., 1985; Bradley and Erickson, 1981; our unpublished results). Using the rate constant for the reaction between dimethyl sulfoxide and OH radical it has been possible to calculate that the OH radical travels an average of 15 ,~ from the site of formation to the reaction center (Ward et al., 1985). This short distance (less than the DNA helix diameter) sug- gests a site-specific reaction. Supporting this view is the observation that the nuclear factor which mediates the damaging action of H202 on DNA was removed when nuclei were dialyzed against EDTA, but not when dialysis was against buffered salt solution (Meneghini and Hoffmann, 1980). Very likely, EDTA removed the iron ion responsible for the Fenton reaction from chromatin.

The in vivo rate of formation of DNA strand

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breaks is not linear with H202 concentration but levels off at a certain H202 concentration range, which depends on the cell type (Hoffmann et al., 1984). One possible explanation is that the limiting factor is the amount of metal bound to chromatin, considering a site-specific Fenton reaction. Different cell types could have distinct extents of iron bound to chromatin which would explain the different levels of strand break saturation. Another possibility is that the limiting factor is the agent which reduces the metal into the form required for the Fenton reaction.

An additional strong indication that a Fenton reaction is responsible for the DNA strand breaks formed in intact cells exposed to H202 comes from experiments with metal chelators. Some of them bind iron rendering the metal incapable of generating OH radicals by a Fenton reaction. Most of them, however, have charged or large structures and cannot diffuse into the cell. Exceptions are the lipophilic compounds o-phenanthroline and ~t,a'-bipyridine which prevent iron from par- ticipating in the Fenton reaction and protect intact cell DNA from the eflects of H202 or a mixture of H202 and O 2 produced by the xanthine oxidase system (Meneghini and Mello- Filho, 1985; Birboim and Kanabus-Kaminska, 1985). That these chelators have to enter the cell in order to exert their effects is shown by the fact that desferrioxamine, a peptide of molecular weight 560, and bathophenanthroline disulfonate, a charged derivative of o-phenanthroline, only poorly prevented the formation of DNA strand breaks in H202-treated cells; by contrast, EPR experiments have shown that these compounds completely abolished iron participation in Fenton reactions (Mello-Filho and Meneghini, 1985). The fact that o-phenanthroline affords protection indicates that iron is the metal involved in this Fenton reaction. This is because copper-o-phenanthroline complexes are even better Fenton reactants than copper alone (Que et al., 1980; Pope et al., 1982). However iron needs to be reduced to react in a Fenton reaction. Although this reduction may be conducted by H202:

H202 + Fe 3+ ---* Fe 2+ + 0 2 + 2 H ÷ (IX)

This is a slow reaction and in fact when nuclei are

exposed to H202 in the absence of a reducing agent only a few strand breaks are formed. The fact that in intact cells H202 produces DNA strand breaks to a much larger extent shows that a cellular reducing agent responsible for the iron redox cycle is required. The Haber-Weiss reaction, in which O 2 is the iron-reducing agent, could explain the whole process, as it occurs in vitro (Mello-Filho and Meneghini, 1985). An indication that this is so comes from experiments in which cells are exposed simultaneously to H202 and diethyldithiocarbamate. This latter compound is an efficient C u / Z n SOD inhibitor (Heikkila et al., 1976) and would be expected to bring about an increase in the intraceUular 0 2 steady-state concentration. This in turn should enhance the Haber-Weiss reaction due to an increase in the rate of Fe 3+ reduction. This expectation was fulfilled when cells were exposed to H202 plus diethyldithiocarbamate (Mello-Filho and Meneg- hini, 1984) which indicates that at high O 2 intracellular concentration the Haber-Weiss reaction is responsible for DNA cleavage. However it remains to be proved whether at normal steady-state concentration O~- is the main Fe 3+ reductant. Ascorbate has been claimed to be a good candi- date for this role (Winterbourn, 1979). A possible approach to test the 0 2 role in DNA cleavage is to increase the SOD activity in the cell, either by microinjection of the enzyme or by transfection of the SOD gene. Both conditions have been worked out but in neither case has the effect of reactive oxygen species on DNA been tested (Bagley et al., 1986; Eroy-Stein et al., 1986). The opposite condition, that of obtaining SOD minus mutant, by gene disruption has only been achieved in yeast (Van Loon et al., 1986) and must be a difficult task in mammalian cells. In Chinese hamster fibroblasts with extra copies of the C u / Z n SOD gene lipid peroxidation was enhanced. The explanation offered was that higher SOD levels brought about a higher H202 steady-state concentration (Eroy-Stein et al., 1986). This explanation is acceptable if 0 2 has no major role in lipid peroxidation either as a direct oxidant or as an iron-reducing agent. Certainly more data are required to determine whether cellular 0 2 repre- sents, in fact, a deleterious species for DNA in terms of its reducing capacity toward Fe 3+, as

opposed to external O~- which is ineffective in terms of DNA damage (Mello-Filho and Meneg- hini, 1985).

It is of great interest to determine the macro- molecular complex of iron responsible for the Fenton reaction which leads to the DNA lesion. Iron binds firmly to DNA (Shires, 1982) and the complex seems to participate in the Fenton reaction (Floyd, 1981), but it has not been established whether this complex is formed in vivo and if it participates in the Fenton reaction. If this is so it is intriguing how iron is allowed to bind such a critical target as DNA. In fact all evidence points to iron being carefully handled by the cell, in general being kept as relatively inert protein complexes (from the viewpoint of the Fenton reaction) before being used in the building up of enzymes and other proteins. Both the transferrin receptor, a membrane protein responsible for iron uptake, and ferritin, an iron storage protein, participate in the transport and availability of cellular iron (Ai- sen and Listowsky, 1980). The recent finding that several classes of proteins involved in nucleic acid binding or gene regulation contain metal-binding domains (Berg, 1981) is also relevant in this context.

Certainly, the Fenton reaction is not the only chemical process responsible for the action of active oxygen species on DNA. When leukocytes are stimulated with phorbol myristate acetate (PMA) they produce 0 2 and its dismutation product H202 (Goldstein et al., 1979). Contrary to the situation where O~ plus H202 are generated externally by the xanthine oxidase system, in the phorbol ester-stimulated leukocytes only 50% of the DNA strand breaks are prevented by catalase; moreover, the remaining 50% are prevented by SOD (Birboim and Kanabus-Kaminska, 1985).

Both these types of strand breaks are repaired more slowly than strand breaks produced by ionizing radiation (Birboim, 1986). It is known that most of the y-ray-induced strand breaks are produced by an OH radical arising from water radiolysis. Therefore it has been argued that the O2-induced breaks (suppressed by SOD) are not brought about by OH radicals (Birboim, 1986). Because in PMA-stimulated leukocytes the H202- induced breaks are also prevented by o- phenanthroline (Birboim and Kanabus-Kaminska,

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1985), it is difficult to understand how OH radicals are not involved in the process. Because 0 2 is not a DNA oxidant it is possible that it participates in some rnebrane or intracellular reaction which produces a DNA oxidant species. In this context it is noteworthy to mention that phorboi ester stimulates the production of clastogenic factors on lymphocyte membranes which are released into the medium and may cause chromosomal alterations in neighboring cells (Emerit and Cerutti, 1982). This process involves lipoperoxida- tion and both in vitro (Inouye, 1984) and in vivo (Ochi and Cerutti, 1987) experiments have shown that lipid hydroperoxides cause double-strand breaks in DNA. As opposed to H202-induced breaks which were blocked by o-phenanthroline, the O~--induced breaks were enhanced by the chelator, indicating that they do not involve a Fenton reaction (Birboim and Kanabus-Kaminska, 1985). In human fibroblasts phorbol esters also induced DNA strand breaks which in this case were prevented by dimethyl sulfoxide and catalase (Snyder, 1985), suggesting mediation by a Fenton reaction. The events that occur in membranes of cells exposed to phorbol esters are likely to be important in carcinogenesis, as will be discussed in the next section.

Several other forms of generating intracellular oxygen radicals have been described. Most of the DNA strand breaks produced by radiation are a consequence of attack by the OH radical formed by water radiolysis (Repine et al., 1981a). Al- though H202 is also produced by ionizing radiation the OH radical from water radiolysis is com- paratively much more important in terms of strand break production. In fact, o-phenanthroline affords no protection to D N A from "y-ray-produced strand breaks (Mello-Filho and Meneghini, 1985). Photochemical reactions induced by cellular chro- mophores, like riboflavin, may lead to the production of 0 2 and H202 and many of the deleterious effects of near-ultraviolet and visible lights are mediated by oxygen species (Hoffmann and Meneghini, 1979a; Parshad et al., 1978; Wang et al., 1980; Erickson et al., 1980; Danpure and Tyrrell, 1976). Several compounds are metabolized with a simultaneous high production of superoxide and other oxygen radicals. Thus for instance the herbicide paraquat (Farrington et al., 1973)

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and many quinones (Nohl et al., 1986) are quickly reduced to a radical form by NADPH. These radicals are good electron donors to molecular oxygen, increasing the steady-state O~- concentration and bypassing the cytochrome oxidase system in the normal electron flux from NADPH to 02. This cytochrome oxidase-independent consumption of oxygen may be measured in the presence of cyanide, a strong inhibitor of this enzyme. In bacteria it has been shown that paraquat produces DNA lesions and induces SOS response via production of O~- (Brawn and Fridovich, 1985). It has also been shown that menadione, a quinone related to vitamin K, produces DNA strand breaks in V79 cells and that this production is inhibited by an OH radical scavenger (E.L. Martins, unpublished results). Interestingly, o-phenanthroline affords only partial protection in the latter case indicating some Fenton-independent production of OH radical via the menadione semiquinone. It is noteworthy that semiqninones are components of cigarette tar, which have recently been shown to promote DNA strand breaks mediated by active oxygen species (Cosgrave et al., 1985).

From the examples cited above it seems advisa- ble to avoid generalizations conceming the production of DNA damage by oxygen species. Al- though Fenton-generated OH radicals (or an equivalent chemical species) do produce DNA lesions in vivo we must keep an open mind to other possibilities. Some important questions are: (i) What metal-reducing agent(s) is important in vivo to render H20 z capable of promoting signifi- cant DNA damage? (ii) Is a transition metal al- ways required to produce OH radical from H202 or can some organic radical (a semiquinone for example) also play such a role? (iii) Although O~- is not a direct DNA oxidant can it generate other cellular species in the absence of H202 which are capable of producing DNA damage (lipid peroxide for example)? (iv) To what extent can different conditions of OH radical generation (e.g. a site-specific versus a non-site-specific Fenton reaction) affect the spectrum of DNA lesions ob- tained. These are not the only but are certainly very important questions which have only pre- liminarily been addressed.

Biological responses to the action of oxygen species on D N A

A universal and immediate response to DNA damage is DNA repair. Given the appreciable specificity of the DNA repair systems and considering the relatively large spectrum of DNA lesions produced by oxygen species, several repair pathways are likely to be elicited when cells are exposed to such species. In mammalian cells these repair pathways are ill-characterized. Most of the repair studies have focused on single-strand breaks which in fact represent a mixture of different DNA structural modifications, including direct phosphodiester breaks, breaks originated from chemical modifications in the deoxyribose rings, breaks which are generated in alkaline conditions by labilization of phosphodiester bonds adjacent to sites that lost the base, and finally breaks corresponding to repair intermediates brought about by repair endonucleases. In spite of this heterogeneity of potential lesions the repair kinetics of single-strand breaks produced by H202 in mammalian cells are a first-order process (Bradley and Erickson, 1981; Wang et al., 1980; Cantoni et al., 1986) as opposed to repair of strand breaks caused by ionizing radiation (Bradley and Erick- son, 1981).

In spite of a first line of defence, constituted by the antioxidants, and of a second line of defence, performed by repair enzymes, lesions produced by active oxygen species may still remain in DNA and produce chromosomal damage, cell killing, mutation and cell transformation. For the sake of clarity I shall discuss these biological endpoints separately.

Loss of cell viability One initial and frequently overlooked problem

is to define cell viability. The capacity of individ- ual cells to form colonies has been subject of many quantitative studies in radiobiology and seems to depend on the integrity of the genome. Exclusion of dyes, release of chromium-51 from loaded cells and release of enzymes are alternative parameters to measure cell viability which are likely to depend on cell membrane integrity. De- pending on the damaging agent employed these structures will be affected in different ways and

one cannot speak generically of cell viability without specifying the parameter considered. In the case of active oxygen species we have noticed that H202 concentrations that render the cells completely unable to form colonies are not sufficient to affect dye exclusion capacity (unpublished results). These cells may thus become non-viable well before their membrane is affected. For clarity I shall refer to survival capacity in terms of colony formation as colony-forming ability (cfa).

In bacteria cfa is affected only at high H202 concentrations. There are two modes of killing, one in the 1-10-#M range which depends on DNA damage and another at higher concentrations, that probably involves other targets. An intermediate concentration range, in which resistance is observed, has been ascribed to the scavenging action of H202 towards OH radical (Imlay and Linn, 1986). It has also been observed that killing of bacteria by H202 increases with iron concentration in the medium, suggesting a mediation by Fenton reaction (Repine et al., 1981b). The observation that sublethal doses of H202 enhance E. coli resistance to subsequent challenge with higher H202 doses (Demple and Halbrook, 1983) has been ascribed to the induction of scavenging enzymes, like catalase (Tyrell, 1985). Enteric bacteria have several enzyme activi- ties that may protect the cells from oxidative damage. A variety of oxidative stresses and heat- shock induce the accumulation of AppppA and a series of related adenylylated dinucleotides (Bochner et al., 1984). These dinucleotides may be alarmones that prepare cellular metabolism to cope with oxidative stresses. An oxy R gene has been described in Salmonella typhimurium which con- trois gene expression related to response to oxidative stress (Christman et al., 1985). This gene seems to produce a protein which is a positive effector of gene expression of at least 9 proteins involved in protection against oxidative stress. It is possible that the alarmones bind the oxy R gene product activating transcription of a specific set of genes.

In mammalian cells cfas are affected distinctly in different cell lines: cells whose DNA undergoes more DNA damage by H202 are those which are killed faster (Hoffmann et al., 1984). The iron chelator o-phenanthroline completely protects the

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cells from the killing effects of H202 or from the products of the xanthine oxidase/xanthine system; moreover, in the latter case SOD affords no protection whereas catalase provides full protection (Mello-Filho and Meneghini, 1985; Mello- Filho et al., 1984). This pattern of killing effects is completely similar to that of the formation of single-strand breaks and again indicates that H202 is the relevant external oxygen species that enters the cell and produces the OH radical in the nucleus by a Fenton reaction. Therefore, the ultimate species to produce the lethal injury in DNA is the OH radical. Bradley and Erickson (1981) measured cfa of HEOE-treacted V79 cells and con- cluded that lethality may be due to damage in targets other than DNA because the survival curves denoted no repair of the lethal damage (i.e., no shoulder in the curves). However, in equivalent assays with the same cells other investigators observed these shoulders (Ward et al., 1985; Hoff- mann et al., 1984). Moreover, fibroblasts from patients with the disease ataxia telangiectasia, which lose cfa at lower doses of "t-ray and lower concentrations of reactive oxygen species, have some defect in the machinery which deals with DNA lesions produced by these agents (Painter, 1981). Therefore, it seems likely that loss of cfa produced by active oxygen species results from lethal injuries to DNA, the nature of which has not yet been established. The concept has been gaining acceptance that single-strand breaks are not lethal (Ward et al., 1985; Bradley and Erick- son, 1981; Cantoni et al., 1986). For instance, Ward et al., (1985) have observed that treatment of V79 cells with 5 × 10 -5 M H202 at 0°C leads to the production of single-strand breaks equivalent to that formed by 10 Gy of "t-ray irradiation. However, effective loss of cfa only occurs at 5 x 10 -2 M, a concentration that in terms of single- strand breaks would correspond to 6000 Gy. They indicate that this favors the idea that DNA single-strand breaks do not constitute toxic lesions and that to produce lethal events "locally multiple damage sites" would be required, like double- strand breaks for example. The production of this lesion requires the generation of at least two OH radicals on the same site. If these radicals are formed by a site-specific Fenton reaction between H202 and ferrous ion bound to DNA, the ferric

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ion can be reduced back to the ferrous form by O2. A new cycle of Fenton reaction may thus occur at the same site, with production of new OH radicals. Because O ; production requires metabolic activity, recycling of iron would only occur efficiently at 37°C and this might explain why loss of cfa at 37°C (Hoffmann et al., 1984) is much more effective than at 0 ° C (Bradley and Erickson, 1981). To test this idea the ratio between double-strand breaks and single-strand breaks produced by H202 at 37°C and 0 ° C could be compared, a much higher value being expected at 37°C.

Exposure of V79 Chinese hamster cells to paraquat also produces loss of cfa (Bagley et al., 1986). Paraquat is known to produce 0 2 in the cell and in these experiments superoxide dismutase introduced in the cell by a "scrape-loading" process provided protection from the killing effect of the chemical; however, catalase introduced in the cell by the same procedure afforded no protection nor did treatment of the cells with 50 mM dimethyl sulfoxide. Based on these data the authors claimed that a Fenton reaction is not mediating the killing effect. However, they failed to show evidence that catalase entered the cell, which is important since diffusion of this enzyme into the cell has been shown to occur very slowly (Stacey, 1977). Moreover, much higher dimethyl sulfoxide concentrations are required to provide protection from OH effects in intact cells (Ward et al., 1985). The lack of protection by desferrioxamine is not a strong argument against the Fenton reaction involvement either, because this iron chelator is a peptide with molecular weight above 500 and has been proven to be ineffective in chelating intracellular iron into a non-redox cycling form as opposed to o-phenanthroline and a,a '-bipyridine, which are low molecular weight liposoluble structures (Mello-Filho and Meneghini, 1985).

Recently Eroy-Stein et al. (1986) succeeded in trasfecting C u / Z n SOD gene into HeLa and mouse ceils. The transfected cells expressed up to 6-fold the normal level of SOD and were more resistant to paraquat than the parental cells, using exclusion of dye as a measurement of viability. This shows that when at a larger excess over the normal level 0 2 has a deleterious effect that can be prevented by an excess of SOD activity. How-

ever, under normal growth conditions they found that transfected cells produced higher levels of lipid peroxidation than parental cells and attrib- uted the fact to increased H202 steady-state concentration. The authors claim that under normal conditions higher levels of SOD activity may be deleterious and assume that this may contribute to some of the clinical symptoms associated with Down's syndrome (whose patients have one extra copy of SOD gene due to the trisomy 21). Unfor- tunately, no assays of cfa have been carried out with these transfected cells. It would be interesting to look at that at normal 0 2 steady state, by exposing cells to H202. If a Haber-Weiss process is involved in the production of the lethal damage (Mello-Filho et al., 1984) a higher SOD activity would be expected to bring about a reduced rate of Fe 3 ÷ reduction and a decreased rate of killing.

It has been shown that macrophages and lymphocytes exposed to H202 undergo single- strand breaks in their DNA (Schraufstatter et al., 1983, 1986). These strand breaks constitute a cofactor for the enzyme poly(ADP-ribose) polymerase to synthesize poly(ADP-ribose) from NAD and as a consequence there occurs a N A D depletion in the cell. Several events follow this depletion, culminating in cell death, measured by the dye exclusion assay. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevents the NAD depletion, the repair of single-strand breaks and prevents also the subsequent events, including cell death. Their explanation for these observations is that DNA strand breaks are not directly responsible for cell lysis. In fact, in the presence of 3-aminobenzamide the cells "survive" even in the presence of a large burden of strand breaks. How- ever, too many breaks would render the cells non-viable. The activation of poly(ADP-ribose) polymerase leads to NAD depletion which ini- tiates a suicidal chain of events, culminating with cell lysis (Schraufstatter et al., 1986). Interestingly, in the case of Chinese hamster ceils 3-aminobenzamide potentiates the H202 cytotoxicity, measured by loss of cfa (Cantoni et al., 1986). However, these observations and those with leukocytes are not mutually exclusive and it would be interesting to see whether in the case of Chinese hamster fibroblasts cell lysis is also mediated by poly(ADP-ribose) polymerase. Accumulation of

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poly(ADP-ribose) mediated by active oxygen species was also observed in mouse and human fibroblast exposed to phorbol myristate acetate (Singh et al., 1985). The authors suggested that the polymer mediates the promoter-induced modula- tion of chromosome structure and gene expression and that this occurs in the absence of single-strand breaks. However, phorbol myristate acetate was shown to elicit single-strand breaks in fibroblast (Snyder, 1985).

Mutations and chromosome alterations Superoxide anion and hydrogen peroxide are

mutagenic in bacteria (Moody and Hassan, 1982). In E. coli paraquat induces SOS responses and the cells are rendered resistant to the lethal effects of ultraviolet light (Brawn and Fridovich, 1985). In mammalian cells earlier experiments failed to show mutagenic action of H202 (Bradley and Erickson, 1981). However, Phillips et al. (1984) observed an increase in the frequency of thioguanine resistance (HGPRT locus) in CHO cells exposed to the xanthine oxidase/xanthine system. Catalase, but not SOD, prevented the induction, indicating that externally generated H202 is the mutagenic agent. We also observed an increase in the frequency of azaguanine resistance in V79 cells exposed to H202 which was prevented when o-phenanthroline was present denoting that a Fenton reaction mediated the process (unpublished results). In addition, leukocytes stimulated to an oxidative burst produced mutations at the HGPRT locus of target fibroblasts (Weitberg et al., 1983). It seems clear that oxygen species are capable of producing mutation and the failure of Bradley and Erickson (1981) to observe mutation may be due to exposure to H202 at 0 °C instead of 37 o C. In fact, the spectrum of DNA lesions at these two temper- atures may be quite different if we consider, as discussed above, that multiple hits at the same site may occur at 37 ° C. However, it seems clear that at equitoxic doses ionizing radiation is more mutagenic than active oxygen species. Certainly, further studies on mutagenesis at specific loci by active oxygen species are required. The importance of these studies may be more emphasized if we consider that stimulated leukocytes may produce mutations in cells at the inflammation site (Weitberg et al., 1983) and that mutagenicity of

organic compounds may be mediated by active oxygen species (Solanki et al., 1984; Monny and Michelson, 1981).

Chromosome aberrations (CA) and sister-chromatid exchanges (SCE) are induced by active oxygen species as determined by many studies. There seems to be a correlation between these events and mutagenesis and carcinogenesis. Induc- tions of CA and SCE have different requirements in terms of active oxygen species. Thus in HeLa cells H202 is sufficient to produce SCE but although necessary is not sufficient to produce CA (Estervig and Wang, 1984). This is in agreement with other studies which have shown that the system xanthine oxidase/xanthine produces both SCE and CA in CHO cells and that they both are prevented by catalase but only CA is prevented by SOD (Philips et al., 1984). It seems that DNA strand breaks produced by H202 may give rise to SCE whereas some other factor is required for CA. Perhaps 0 2 is important because it participates in the formation of lipid peroxides in the membrane which could be somehow involved in the production of CA. This is in the direction of the idea that O~- is important for the production of clastogenic factors (factors that cause chromosome alterations) in the cell membrane (Emerit and Cerutti, 1982; Emerit et al., 1985; Ochi and Cerutti, 1987). These clastogenic factors are formed when lymphocytes are exposed to a source of O~- production or when they are treated with the cancer promoter phorbol myristate acetate (PMA). A chain of reactions involving stimulation of arachidonic acid cascade and lipid peroxidation follows and clastogenic factors are formed and released into the medium. It has been proposed that the etiology of Bloom's syndrome, a disease whose patients develop malignancies at a much higher frequency than normal individuals and whose cells exhibit a high frequency of CA and SCE, resides in a deficiency in the detoxification of active oxygen species (Emerit and Cerutti, 1981). In fact, cocultivation of Bloom's syndrome with other cell types affects the frequency of CA in all the cells. The addition of SOD to cultures of phytohemagglutinin-stimulated lymphocytes from normal individuals suppresses the clastogenic activity present in concentrated ultrafiltrates of Bloom's syndrome cell media (Emerit and Cerutti,

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1981). In this context, an elevated level of SOD has been detected in Bloom's syndrome cells (Nicotera et al., 1985), although this fact is not immediately reconciled with the aforementioned model.

The mechanisms of SCE and CA production by active oxygen species remain to be established. The production of SCE by H202 or by the xanthine oxidase/xanthine system in V79 cells is completely prevented by o-phenanthroline (Larra- mendy et al., 1987). The iron chelator has no effect on the production of SCE by the alkylating agent methyl methanesulfonate. These observations are in agreement with SCE generated by active oxygen species being promoted by DNA lesions formed in a Fenton type of reaction.

Carcinogenesis

The use of transformation of cells in culture has been of paramount importance as an in vitro model for carcinogenesis. Although yet a complex system it has been possible to dissect in vitro malignant transformation into some operationally defined steps, in analogy to in vivo carcinogenesis. Two of these steps are initiation and promotion. It has become widely accepted that initiation of cell transformation involves mutagenic events in genes responsible for control of cell growth (Guerrero et al., 1984; Marshall et al., 1984). Promotion, on the other hand, is the phenomenon of enhancement of transformation of previously initiated ceils by agents which per se are not necessarily carcino- genic, like PMA. The importance of active oxygen species in both initiation and promotion has become increasingly appreciated in recent years (Ames, 1983). One very important discovery was that certain antioxidants can prevent the promotion process in several cell transformation systems (Solanki et al., 1984; Goldstein et al., 1981; Kensler et al., 1983; Kennedy et al., 1984; Borek and Troll, 1983; Nakamura et al., 1985; Armato et al., 1984). In addition, free radical-generating systems are promoters of cell transformation (Slaga et al., 1981; Zimmerman and Cerutti, 1984; Weitzman et al., 1985). However, the mechanisms of promotion are rather unclear and there has been dispute as to whether promoters affect DNA or not (Marx, 1983). Also unclear is which oxygen species are

involved in tumor promotion. SOD inhibited promotion by PMA in JB6 mouse epidermal cells (Nakamura et al., 1985) and in hamster embryo cells (Borek and Troll, 1983), whereas catalase did not. Exactly the opposite results were observed in C3H 10T1/2 mouse fibroblasts (Kennedy et al., 1984). In these same cells the promotion effect was achieved by treating the cells with the xanthine oxidase/xanthine system instead of PMA and in this case both SOD and catalase suppressed the effect, indicating the involvement of O~- and H 202 (Zimmerman and Cerutti, 1984). Perhaps SOD suppression of promotion by PMA was not achieved because under these conditions active oxygen species are generated in the membrane moiety, where SOD has no access, at the same time that 0 2 is not released into the medium to be destroyed by SOD. As opposed to that the SOD-like liposoluble compound copper diisopro- pylsalicylate, did prevent promotion induced by PMA (Kensler et al., 1983; Kennedy et al., 1984). However, it should be remembered that it remains to be proven that this copper complex is suppress- ing the tumor promotion by its SOD-like activity.

At any rate it seems clear that active oxygen species mediate the phenomenon of tumor promotion. As mentioned before, leukocytes exposed to PMA undergo DNA strand breaks (Birboim and Kanabus-Kaminska, 1985) and release clastogenic factors (Emerit and Cerutti, 1985). It is attractive, therefore, to think that there might be a link between the inflammatory properties of PMA and the high cancer incidence in certain chronic inflammations. The clastogenic factors might establish this link if promotion indeed is mediated by chromosomal alterations. However, there is no straightforward relationship between inflammatory agents and promoters. Moreover, the promotion effect of PMA is observed in transformation of fibroblasts in culture and in these cells it is not clear whether PMA induces O~- production and clastogenic factors (Kinsella et al., 1983) although strand breaks have been detected in human diploid fibroblast exposed to PMA (Snyder, 1985). Nevertheless, when leukocytes are stimulated to a state of oxidative burst by PMA and other promoters, target cells undergo both chromosomal damage (Weitberg et al., 1983) and cell malignant transformation (Weitzman et al., 1985). Therefore

it is possible that the in vivo act ion of promoters is mediated by the oxidative burst of leukocytes and that the relevant molecular events consist of D N A damages and the ensuing chromosomal arrangements. In this context promoters , a l though not mutagenic per se, would act as a leukocyte- mediated mutagenic agent (Frenkel et al., 1986). The correlation between chronic inf lammat ion and cancer is stressed by the known ant ipromotional properties of ant i - inf lammatory drugs (Belman and Troll, 1972).

Al though emphasis has been put on the act ion of active oxygen species on tumor promot ion, several studies dealt with their involvement in tumor initiation as well. In fact, ant ioxidants such as selenium and vi tamin E inhibit in vitro C3H cell t ransformat ion induced by X-ray, benzo[a]- pyrene and t ryp tophan pyrolysate (Borek et al., 1986). Selenium induced an enhancement of the glutathione levels, whereas vi tamin E is known to inhibit peroxidation. Both neurophils st imulated to synthesize active oxygen species or the xanthine ox idase /hypoxan th ine system can induce the whole in vitro malignant t ransformat ion in C3H cells (Weitzman et al., 1985). It has also been shown that initiation by d imethylbenz[a]anthracene is mediated by oxygen species (Solanki et al., 1984). Thus, active oxygen species are involved in bo th initiation and promot ion of tumors.

The mechanisms of initiation and p romot ion of mal ignant t ransformat ion only recently have become amenable to a more promising investigation. Certaintly, the knowledge of the mechanisms whereby active oxygen species part icipate in those processes must await further impor tant dis- coveries. Nevertheless, the knowledge that oxygen species are clearly involved in carcinogenesis is per se of u tmost importance. These species are normal metaboli tes in the cells where their levels can be changed by oxidants and antioxidants present in diet and in the general environment (Ames, 1983). Certainly new epidemiological and experimental da ta will in the near future provide us with a bet ter unders tanding of mechanisms and with possibilities of control of genotoxic effects of active oxygen species.

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Acknowledgements

This work was supported by grants f rom FAPESP, C N P q and F I N E P , Brazilian agencies of science. I thank Dr. Lilian Nassi, Alber to Mello- Fi lho and Elizabeth L. Mart ins for reviewing the manuscr ipt and Miss A. Virginia M. Claure for typing the manuscript .

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genotoxicity of active oxygen species in mammalian cells

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