Carcinogen-mediated oxidant formation and oxidative DNA damage

Pharraac. Ther.Vol. 53, pp. 127-166, 1992 Printed in Great Britain. All rights reserved

0163-7258/92$15.00 © 1992PergamonPress Ltd

Associate Editor: D. GRUNBERGER

CARCINOGEN-MEDIATED O X I D A N T FORMATION A N D OXIDATIVE D N A D A M A G E KRYSTYNA FRENKEL Departments of Environmental Medicine Pathology, Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, N Y 10016-6451 U.S.A.

Abstract--This article reviews the experimental data that points to formation of reactive oxygen species (ROS) and oxidative DNA base damage as being important contributors to cancer development. Particular emphasis is placed on the role they play in genetic changes occurring during tumor promotion. A number of structurally different anticarcinogenic agents inhibit ROS production and oxidative DNA damage as they inhibit inflammation and tumor promotion. This underlines the importance of ROS and oxidative genetic damage to the carcinogenic process. It also points to the possibility that some types of cancer may be preventable if the cycles of tumor promotion can be interrupted. CONTENTS 1. Introduction 2. Formation of Reactive Oxygen Species (ROS) by Tumor Promoter-mediated Processes 2.1. Oxidative burst by polymorphonuclear leukocytes (PMNs) and macrophages 2.1.1. Agents activating PMNs and macrophages 2.2. Oxidant formation by non-phagocytic cells 3. Carcinogen-mediated ROS Formation and Utilization 3.1. ROS produced by ionizing and u.v. radiation 3.2. ROS induced by chemical carcinogens 3.2.1. ROS formation due to metabolism of carcinogens by mixed-function oxidase 3.2.2. ROS utilization by peroxidatic oxidation of carcinogens 3.3. ROS formation mediated by carcinogens that do not bind to DNA 3.4. ROS induction by metal carcinogens 4. Other Sources of ROS 4.1. Endogenous sources 4.2. Exogenous sources 5. Oxidative DNA Damage in vitro 5.1. Damage caused by ionizing radiation 5.1.1. Thymine moiety: Formation of thymine glycol (TG) and 5-hydroxymethyl uracil (HMU) 5.1.2. Other DNA bases: Formation of 8-hydroxyl(oxo)guanine (8-OHG) 5.1.3. Analysis of DNA: Quantitation of oxidized DNA bases 5.2. Damage caused by tumor promoters 5.2.1. Formation of TG, HMU, 5-formyl uracil and 8-OHG in DNA

128 131 131 132 134 135 135 136 136 137 137 138 138 138 139 139 139 139 141 141 142 143

Abbreviations--ROS, reactive oxygen species; "O~-,superoxide anion radicals; H202, hydrogen peroxide; 'OH, hydroxyl radicals; IO2, singlet oxygen; HOCI/OCI-, hypochlofite; HMU, 5-hydroxymethyl uradl; TG, thymine glycol; FPU, N'-formyl-N-pyruvylurea; HMH, 5-hydroxyl-5-methylhydantoin;8-OHG, 8-hydroxyl(oxo)guanine;FapyG, 2,6-diamino4-hydroxy-5-formamidopyrimidine;FapyA, 5-formamido-4,6-diaminopyrimidine;dTG, thymidine glycol; HMdU, 5-hydroxymethyl-2'-deoxyufidine; FdU, 5-formyl-2'-deoxyuridine; 8-OHdG, 8-hydroxyl(oxo)-2'-deoxygunnosine;HPMdU, 5-hydroperoxymethyl-2'-deoxyufidine; PAHs, polycychc aromatic hydrocarbons; B(a)P, benzo(a)pyrene; B(e)P, benzo(e)pyrene; DMBA, 7,12-dimethylbenz(a)anthracene; 3MC, 3-methylcholanthrene; DES, diethylstilbestrol; HPLC, high pressure (performance) liquid chromatography; GC-MS, gas chromatography coupled with spectroscopy; ELISA, enzyme-linked immunosorbent assay; HMdU-BSA, HMdU covalently hound to bovine serum albumin; PMNs, polymorphonuclear leukocytes; MPO, myeloperoxidase;SOD, superoxide dismutase; TPA, 12-O-tetradecanoyl-phorbol-13acetate; RPA, 12-O-retinoyl-phorbol-13-acetate;EGCG, ( - ) . epigallocatechin gallate; CAPE, caff¢icadd phenethyl ester; GSH, glutathione; CuDIPS, Cu(3,5-diisopropylsalicylate)2.

127

K. FRENKEL

128

5.3. Damage caused by chemical carcinogens 5.3.1. Oxidative DNA modification by benzo(a)pyrene [B(a)P] 5.3.2. Oxidative DNA modification by other carcinogens 6. Oxidative DNA Damage in vivo and its Prevention 6.1. DNA damage induced by tumor promoters 6.1.1. 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced formation of TG, HMU and 8-OHG 6.1.2. Comparison of TPA effects with those of mezerein and 12-O-retinoyl-phorbol-13-acetate (RPA) 6.1.3. Effect of anti-tumor promoters on TPA-mediated DNA damage 6.1.4. TPA-induced DNA modification in epidermis of SENCAR versus C57BL/6J mice 6.2. Carcinogen-mediated formation of oxidized DNA bases 6.2.1. By inorganic carcinogens 6.2.2. By 7,12-dimethylbenz(a)anthracene (DMBA) and B(a)P 6.2.3. By other organic carcinogens 6.3. Oxidative DNA damage in humans 7. Role of ROS and Oxidative DNA Damage in Carcinogenesis 7.1. Role of ROS in cell transformation 7.1.1. Oxidative DNA damage and cell transformation 7.2. Possible participation of abnormal phagocytes in carcinogenesis 7.3. ROS, oxidized DNA bases and oncogene expression 8. Conclusions Acknowledgements References

144 144 144 145 145 145 145 146 146 147 147 147 148 148 149 149 150 150 150 152 153 154

1. I N T R O D U C T I O N The classical two-stage experimental carcinogenesis model has been operationally divided into initiation and promotion stages (Boutwell et al., 1982; Perera, 1991). The initiation step occurs when for example an adduct is formed between an agent (used at a subcarcinogenic dose) and a D N A base, which leads to a mutation (Lutz, 1979; Miller and Miller, 1981; Ashurst et al., 1983; Bigger et al., 1983; DiGiovanni et aL, 1986). This process may result in tumors, when it is followed by multiple applications of a tumor promoter, which can occur even a long time after the initiator (Fig. 1A). Promotion is more complex and much more controversial. T u m o r promoters are thought to induce pleotropic epigenetic changes that lead to clonal expansion of the initiated cells (Slaga et al., 1982; Perchelet and Perchelet, 1988). However, it becomes more apparent that the order of experimental application of promoters is not that important. When the promoter is applied before the initiator (Fig. 1B), it causes cellular changes that are remembered by the cells if followed by an initiator within about 8 weeks (FiJrstenberger et al., 1983). This brings us to the operational distinction between types of tumor promoters. Currently, they can be divided into first- and

A

COMPLETE TUMOR PROMOTER

I PROMOTER I ~INITIATION INITIA~ON__~ I STAQE (Conversion)

I STAGE. PROMOTER (Clonsl Expansion)

PROGRESSIO~ N

CANCER

t

Stagesatwhichgenetic events are likely to involveROSand/orfreeradicals.

B J STAGE I PROMOTER d

L J-L (ClonslJ

(conwmo.) -']-I INITIATIONt

~r,Gs. PROMOTE. I'--J Expansion) ~

PROGRESSION--~CANCER

Stagesatwhich genetic events are likely to involveROSand/orfree radicals. F1o. 1. Simplified model of multi-stage carcinogenesis. Arrows point to stages at which genetic events are likely to involve ROS and/or free radicals.

Carcinogen-mediated oxidant formation and oxidative DNA damage

129

second-stage tumor promoters, as well as so called complete tumor promoters (Slaga et aL, 1980; Fiirstenberger et al., 1981; Kinzel et al., 1986). The first stage promoters are often referred to as convertogenic because they are thought to induce a genetic change that is heritable for short periods of time (Kinzel et aL, 1986). The second stage promoters are assigned functions that previously were considered to be a property of tumor promoters in general, that is clonal expansion (propagation). Hence, complete tumor promoters possess both types of properties: convertogenic and mediating clonal expansion (propagation). There is also a third stage, tumor progression, which causes transformation of benign into malignant and metastatic tumors (Fig. 1). In the past, tumor promoters were frequently referred to as non genotoxic carcinogens. However, there is accumulating evidence that tumor promoters can induce processes that actually lead to genetic changes (Birnboim, 1983; Troll et al., 1984; Dutton and Bowden, 1985; Lewis and Adams, 1985; Snyder, 1985; Floyd et al., 1986; Frenkel et al., 1986b; Frenkel and Chrzan, 1987a,b; Wei and Frenkel, 1991a). It appears therefore that the main difference between initiating and promoting carcinogens in causing DNA damage is the mechanism by which they induce genetic modification. Many non direct acting carcinogens require metabolic activation before they are capable of interacting with nucleic acids (Jeffrey et al., 1977; Lutz, 1979; Philips et al., 1979). Tumor promoters on the other hand induce cellular processes, which produce intermediates that in turn can cause genetic damage (Frenkel, 1989a). Prominent among these intermediates are reactive oxygen species (ROS). It seems that ROS play a role in initiation, first stage (conversion) tumor promotion and progression (Troll et al., 1982; Cerutti, 1985, 1989; Kensler et al., 1987; Marnett, 1987; Pryor, 1987; Perchelet and Perchelet, 1988). Ionizing radiation is an example of a ROS generating insult, which can act as both an initiator and a promoter (Little and Kennedy, 1982; Jaffee and Bowden, 1986). The primary oxygen radical species generated by the radiolysis of water are hydroxyl radicals (.OH), but in the presence of oxygen, superoxide anion radicals (.O~-) and hydrogen peroxide (H~O2) are also produced (Scholes, 1983; von Sonntag, 1987; Demple and Levin, 1991). The same ROS ('05 and H~O2) can also be formed by a number of normal biochemical pathways (Fantone and Ward, 1982; Freeman and Crapo, 1982; Vuillaume, 1987). However, some of those normal pathways can be subverted by xenobiotics, which would induce excessive or untimely ROS production (Frenkel et al., 1988; Perchelet et al., 1988; Frenkel, 1989a; Trush and Kensler, 1991a,b). Although many anti oxidant defenses are present in normal cells, their effectiveness is also often compromised by ROS. Before reviewing the damaging properties of ROS, it is necessary to mention some of the normal cellular biochemical processes that are sources of ROS (see Table 1). One of the most important utilizations of ROS is during respiration carried out by the mitochondrial electron transport, which starts with molecular O: being sequentially reduced by four electrons into water (Chance et al., 1979; Freeman and Crapo, 1982; Cross et al., 1987). Normally, there is practically no leakage of partially reduced oxygen species. Even if some ROS do escape the tight controls, antioxidant

TABLE 1. Normal Processes that Produce R O S 1. Respiration --Mitochondrial electron transport --Hexose monophosphate shunt 2. Metabolism of xenobiotics --Microsomal electron transport (cytochromes P450 and bs) and mixed function oxidase --Peroxidatic oxidation (MPO, prostaglandin H synthase) 3. Activation of phagocytic cells by natural stimuli --Peripheral blood PMNs, basophils and monocytes --Tissue macrophages --Kupfer cells (liver) --Clara cells (lung) 4. Biosynthetic and biodegrading processes --Arachidonic acid metabolism --Fatty acid CoA oxidases --D-amino oxidase --L-ct-hydroxyacid oxidase --Urate oxidase --Tyrosine peroxidase

130

K. FRENKEL

TABLE2. Normal Antioxidant and Repair Defenses 1. Antioxidant enzymes --Superoxide dismutase ---Catalase --GSH peroxidase ---GSH reductase --GSH-S-transferases 2. Antioxidant proteins --Ceruloplasmin --Transferrin --Lactoferrin --Albumin --Haptoglobin 3. Antioxidant, low molecular weight substances --GSH, NAD(P)H --Ascorbate, urate --~-Tocopherol --fl-Carotene 4. DNA repair enzymes a. Glycosylases acting on: --5-hydroxymethyl cytosine --HMU --TG and products derived from TG (5-hydroxyi-5-methylhydantoin,N'-formyl-N-pyruvylurea, urea) b. Endonucleases c. Poly(ADP)ribose transferase 5. Oxidized protein-degrading enzymes --Macroxyproteinase (MOP)

enzymes and substances, such as glutathione (GSH) or uric acid, protect the other organelles from oxidative damage. ROS also are generated by the microsomal electron transport system and by arachidonic acid metabolism (Chance et al., 1979; Capdevila et al., 1981; Freeman and Crapo, 1982). Another very important source of ROS are the phagocytic cells polymorphonuclear leukocytes (PMNs) and macrophages (Badwey and Karnovsky, 1980; Klebanoff, 1980; Fantone and Ward, 1982; Babior, 1984; Cross et al., 1987; Frenkel, 1989a; Weitzman and Gordon, 1990). These are cells that produce large amounts of ROS in their microbiocidal and tumoricidal capacities. Although much smaller amounts of H202 are generated during thyroid hormone biosynthesis, there would be no iodination of thyroglobulin by tyrosine peroxidase without H202 (Deme et al., 1985). Similarly, functioning of other enzymes such as monoamine oxidase, galactose oxidase, cyclooxygenase and lipoxygenase would not be possible (Cross et al., 1987). Another important source of H202 are peroxisomes, which produce one H202 per each two carbon fragment of the metabolized dietary fatty acids (Freeman and Crapo, 1982; Reddy and Lalwani, 1983; Cross et al., 1987). Since substantial amounts of ROS are continuously produced and utilized, mammalian cells elaborate extensive antioxidant defense and repair mechanisms (Ames et al., 1981; Fantone and Ward, 1982; Ames, 1983; Halliwell and Gutteridge, 1986; Tan et al., 1986; Cross et al., 1987; Ketterer et al., 1987; Ketterer, 1988; Vuillaume, 1987; Perchelet and Perchelet, 1988), examples of which are given in Table 2. These mechanisms have been the subject of separate reviews and, therefore, will be presented here only marginally. Those defenses consist of: (1) Antioxidant enzymes (superoxide dismutase (SOD), catalase, GSH peroxidase, reductase and S-transferases), (2) antioxidant proteins, (3) antioxidant low molecular weight substances (GSH, uric acid, ascorbic acid, ~t-tocopherol and fl-carotene), and, if the antioxidants fail and some oxidation of DNA does occur, (4) repair enzymes (N-glycosylases recognizing oxidized bases in DNA, such as thymine glycol (TG), 5-hydroxymethyl uracil (HMU) and 5-hydroxymethyl cytosine, as well as endonucleases and poly(ADP)ribose transferase (Sugimura and Miwa, 1983; Hollstein et al., 1984; Lunec, 1984; Doetsch et al., 1986; Berger et al., 1987; Boorstein et al., 1987a,b; Higgins et al., 1987; Cannon et al., 1988; Teebor et al., 1988; Wallace, 1988; Breimer, 1990). Nuclear DNA is also protected from oxidative damage by its localization distant from the major sources of ROS as well as by proteins of chromatin. In this review, I will present some pathways that are known to contribute unnecessary or excessive ROS, such as phagocytic cells stimulated by tumor promoters, metabolism of carcinogenic

Carcinogen-mediated oxidant formation and oxidative DNA damage

131

xenobiotics and induction of ROS producing enzymes. However, the main emphasis will be on the genetic damage that is caused by those ROS. 2. FORMATION OF REACTIVE OXYGEN SPECIES (ROS) BY TUMOR PROMOTER MEDIATED PROCESSES 2.1. OXIDATIVEBURSTBY POLYMORPHONUCLEARLEUKOCYTES(PMNs) AND MACROPHAGES PMNs are immune cells whose normal function is to recognize, phagocytize and destroy invading bacteria (Badwey and Karnovsky, 1980; Klebanoff, 1980; Fantone and Ward, 1982; Freeman and Crapo, 1982; Babior, 1984; Cross et al., 1987; Warren et al., 1987). When encountering bacteria or other opsonized particles, PMNs respond with a respiratory burst, of which the oxidative burst is an important bacteriocidal part. However, they can also be activated by other unnatural stimuli such as some allergens and tumor promoters. Regardless of the stimulus, the oxidative burst is characterized by a rapid consumption of molecular oxygen followed by an almost concomitant (within 1-2 min) production of substantial amounts of .O~-. Formation of .O~- from molecular oxygen is catalyzed by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Babior, 1984, 1987; Warren et al., 1987) that uses NADPH as the reductant (eqn 1): NADPHoxidase

02 + NADPH •

, "O~-+ H + + NADP +

(1)

NADPH is regenerated by the action of hexose monophosphate shunt enzymes, which utilize glucose as the substrate. At neutral pH, superoxide exists predominantly as .O~-. However, under acidic pH, it exists as HO2, which is in equilibrium with "02 at pH 4.8. Spontaneous or enzymatic (SOD) dismutation of .O~- results in the formation of H202 (eqn 2), with rate constants of 8 x 104 M- t sec- ~ at pH 7.8 or 2 x 109 M- 1see- I, respectively (Fridovich, 1978; Aust et al., 1985). 2"02 + 2H+-*H202 + O5 (or tO2) (dismutation)

(2)

H202 is the precursor of the actual bacteriocidal species .OHs, hypochlorite (HOC1/OC1-) and singlet oxygen (IO2) (eqns 4-7). Hydroxyl radicals are generated from H202 and .O~- in a series of reactions referred to as the iron catalyzed Haber-Weiss reaction (eqn 5). In this reaction Fe(III) is reduced by .O~- to Fe(II) (eqn 3), which in turn reduces H202 to "OHs, a very potent oxidant. This last reaction is often called the Fenton reaction. Fe(III) + "02 ~ Fe(II) + O 5

(3)

Fe(II) + H202--*'OH + O H - Fe(III) (Fenton)

(4)

F¢

•02 + H202

, "OH + OH- + O5 (Haber-Weiss)

(5)

The same reaction can be catalyzed by reduced copper ions and by the chelates of both Fe and Cu (Aust et al., 1985; Halliwell and Gutteridge, 1986). Some carcinogenic metal derivatives appear to catalyze the Fenton reaction when complexed with appropriate ligands, such as Ni(II) bound to the histidine moiety of small oligopeptides (Inoue and Kawanishi, 1989; Nieboer et aL, 1989) or histidine alone (Datta et al., 1991; Kasprzak, 1991). Hypochlorite is formed by oxidation of C1- by H202 in a myeloperoxidase (MPO)-catalyzed reaction (Stelmaszyflska and Zgliczyfiski, 1974; Slivka et al., 1980; Weiss et al., 1982, 1983; Babior, 1984; Grisham et al., 1984; Warren et al., 1987) (eqn 6): e l - + H202

MPO , OCl- + H20

(6)

MPO is released from lysosomal granules of PMNs when they undergo an oxidative burst (Babior, 1984, 1987; Warren et al., 1987). The amount of enzyme released depends on the stimulus utilized. For example, a phagocytic stimulus, such as opsonized zymosan, causes release of substantial amounts of MPO, whereas non phagocytic stimuli (such as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) mediate only a low release of MPO from PMNs (Weiss et al., 1982). Hypochlorite reacts readily with primary amines and forms a long lived and very powerful group of the oxidants mono and dichloramines (RNHC1 and RNC12, respectively), which can

132

K. F~NKEL

inactivate lysosomal enzymes and degrade leukotrienes (Stelmaszyfiska and Zgliczyfiski, 1974; Voctman et al., 1981; Thomas et al., 1982; Weiss et aL, 1982, 1983; Henderson and Klebanoff, 1983; Warren et al., 1987). When H202 is present in excess, it acts as a reductant and reduces OCI- back to CI- but with the evolution of a singlet oxygen (Fantone and Ward, 1982) (eqn 7): OC1- + H202---~C1- + H20 + I o 2

(7)

Out of all of the ROS generated during the oxidative burst of PMNs only H20: can readily cross plasma and nuclear membranes and reach DNA (Freeman and Crapo, 1982). Other ROS are either too reactive (i.e..OHs and OC1-) or charged and require anion channels (.O~-), or are soluble in the lipids of the membranes (105) (Freeman and Crapo, 1982; Aust et al., 1985; Halliwell and Gutteridge, 1986). Moreover, in some cases there is either no H202 formed from the substantial amounts of .O2 or not all of the .O~- is dismutated to H202 (Saito and Tomioka, 1979; Pick and Keisari, 1981). Hence, production of .O2 does not necessarily predetermine formation of the equivalent H202 and other ROS. It is thus H202, which by virtue of being neutral and quite unreactive in the absence of reduced transition metal ions, that reaches the nucleus where it can cause site specific damage. It is thought that iron ions bound to the phosphate groups of nucleic acids, or copper ions bound to proteins, can reduce this incoming H202 to .OHs or .OH-like species and that this second generation of ROS is responsible for oxidation of bases and for strand breaks in DNA (Cross et al., 1987; Chevion, 1988; Frenkel, 1989b). In addition to PMNs, stimulated macrophages can undergo an oxidative burst, which is similar to that of PMNs but with an important exception. Although monocytes and immature macrophages contain MPO, they lose this enzyme upon maturation. Hence, upon stimulation, .O~- and H202 can be formed by alveolar and other tissue macrophages, as well as .OHs when in the presence of reduced transition metal ions, but hypochlorite or chloramines cannot be formed (Van Furth et al., 1970; Nakagawara et al., 1981; Babior, 1984; Trush and Kensler, 1991b). The reason for the loss of MPO is not known, however, it is possible that in order to live long macrophages cannot be exposed to the highly destructive hypochlorite. 2.1.1. Agents Activating P M N s and Macrophages

It has been known for a long time that chronic inflammation exerts co-carcinogenic effects (Dunham, 1972; Fantone and Ward, 1982; Rubio and Nylander, 1982; Dolberg et al., 1985; Chester et al., 1986; Weitzman and Gordon, 1990), however, the mechanism by which it contributes to cancer has not been as yet elucidated. The current thought is that during prolonged inflammation ROS generated by activated phagocytes causes incessant damage to previously normal neighboring cells (Frenkel, 1989a; Weitzman and Gordon, 1990). Although those neighboring cells elaborate formidable antioxidant defenses (Table 2) (Teebor et al., 1988; Perchelet and Perchelet, 1989), eventual and irreversible oxidative damage occurs, which may lead to the development of tumors. Exogeneous agents that activate phagocytes in the absence of natural stimuli also induce formation of copious amounts of ROS that have no positive function to perform (such as destruction of invading bacteria and growth of tumors), but cause oxidative damage to neighboring cells (Birnboim, 1982, 1983; Dutton and Bowden, 1985; Lewis and Adams, 1985; Frenkel and Chrzan, 1987b; Wei and Frenkel, 1991a), some of which seem to be heritable (Shirnam6 Mor+ et al., 1987; Boorstein and Teebor, 1988). This point will be discussed in greater detail later on. It appears that one of the hallmarks of the activity exerted by tumor promoters is their ability to induce PMNs to undergo an oxidative burst (Table 3). The most extensively studied promoters are the phorbol ester type tumor promoters of which TPA is the most potent (Goldstein et al., 1981; Frenkel and Chrzan, 1987a; Witz et al., 1987; Sirak et al., 1991; Robertson and Oberyszyn, 1991; Zelikoff et al., 1991). TPA is often referred to as phorbol myristate acetate (PMA). TPA, which is a complete tumor promoter (Fiirstenberger et al., 1981; Slaga et al., 1982), was shown to activate human PMNs, as well as PMNs and macrophages from various animal species, including guinea pigs, rabbits, rats, mice and fish. The majority of these experiments were carried out in vitro, but some (Robertson and Oberyszyn, 1991; Sirak et al., 1991) were performed on cells obtained from the tumor promoter-treated mice.

Carcinogen-mediated oxidant formation and oxidative DNA damage

133

TABL~3. Sources of Pathogenic ROS Endogenous

1. Phagocytic cells stimulated by tumor promoters through: --Protein kinase C activation (TPA, mezerein, RPA, teleocidin, thapsigargin) --Phosphatase inhibition (okadaic acid, palytoxin) --Unknown mechanism (Ni3S2, NiS2, NiS, CdS, CaCrO4) 2. Non-phagocytic cells (epidermal keratinocytes, HeLa, MRC5, 10T1/2 and others) --Treated with tumor promoters --Metabolizing complete carcinogens [PAHs, nitro- and amino-polyaromatics, diethylstilbestrol (DES), metals (Cr, Ni, Hg, Cu) 3. Quinone-semiquinone redox cycling 4. Induction of pro-oxidant enzymes --Xanthine oxidase 5. Inhibition of antioxidant enzymes 6. Induction of fatty acid CoA oxidases by peroxisome proliferaters 7. Ischemia/reperfusion Exogenous 1. Ionizing radiations (~, X-ray, 3H, u.v.) 2. Cigarette smoke 3. Chewing betel nuts 4. Ozone 5. Quinone antibiotics, chemotherapeutic agents, pesticides

Stimulation of human PMNs with phorbol ester type tumor promoters results in the production of ROS, as measured by formation of .O~- and H202 (Table 3). It is this formation of H202 that appears to be related to the in vivo effectiveness of tumor promoters (Frenkel and Chrzan, 1987a; Frenkel, 1989a). For example, when human PMNs were incubated with TPA, mezerein or 12-O-retinoylphorbol-13-acetate (RPA), all three agents activated PMNs and H202 was produced in a time- and dose-dependent manner. However, the highest levels of H202 were generated due to the action of the potent complete tumor promoter TPA, followed by mezerein and the lowest levels by RPA, which is the same order as that of their in vivo potencies. In retrospect, it is not surprising because H202 itself is a tumor promoter. Analysis, of the DNA that was included in these experimental mixtures containing PMNs and tumor promoters, showed that the oxidized thymines TG and H M U were present in amounts proportional to the generated H~O2. Therefore, the levels of TG and H M U also reflected the in vivo potency of tumor promoters used for PMN activation. Recently, similar findings were reported for in vivo treatment of mice with TPA type tumor promoters. PMNs isolated from TPA treated mice generated the highest levels of H202 followed by other agents in order of their in vivo effectiveness (Sirak et al., 1991). Non-TPA type tumor promoters (Kano et al., 1987; Suganuma et al., 1988, 1989) were also shown to activate PMNs and these include okadaic acid and palytoxin (Table 3). The mechanism of PMN activation is probably different depending on the type of tumor promoter used. For example, the TPA type promoters are thought to cause activation of protein kinase C (Nishizuka, 1984; Arcoleo and Weinstein, 1985; Blumberg, 1988), which precedes transformation of the dormant N A D P H oxidase into the active enzyme (Babior, 1987). Whereas the non-TPA type seem to act through inhibition of membrane bound phosphatases (Sassa et al., 1989; Cohen and Cohen, 1989; Matsunaga et al., 1991). Suppression of the phosphatase activity leads to unchecked phosphorylation by the kinases, with the end result being about the same as when kinases themselves are activated. Although it is not known which of the phosphorylated targets is the contributor to the tumor promotional processes, phosphorylation is thought to be necessary. Other non-TPA type tumor promoters, such as benzoyl peroxide and some anthron derivatives (i.e. anthralin and chrysarobin), also do not interact with protein kinase C, a phorbol ester receptor (Blumberg et al., 1984; DiGiovanni et al., 1987). Although by different mechanisms, these tumor promoters can generate free radicals and ROS as do other tumor promoters (DiGiovanni et al., 1988; Trush and Kensler, 1991a,b). In addition to organic tumor promoters, other agents such as carcinogenic metal derivatives are also capable of PMN activation (Table 3) (Klein et al., 1991). These include crystalline Ni3S2, NiS2, NiS and CdS (Zhong et al., 1990) and CaCrO4 (unpublished data), whereas their soluble salts are

134

K. FRENKEL

ineffective. The mechanism by which particulate carcinogenic metal salts elicit activation of PMNs is not clear. However, it cannot be solely due to phagocytosis, because other particulate but non-carcinogenic salts do not stimulate PMNs. Ni3S2 and CdS are known to accumulate in the lungs of exposed populations and are chemotactic for PMNs (Cross et al., 1979; Katsnelson and Privalova, 1984; Lynn, 1984; Sunderman, 1984). Therefore, their ability to stimulate PMNs and to induce production of H202 and other ROS in an exposed target site is likely to contribute to the carcinogenicity of these compounds. 2.2. OXIDANT FORMATIONBY NON-PHAGOCYTICCELLS With the increasing availability of highly sensitive assays, more data are forthcoming that show the formation of oxidants in cells other than the professional phagocytes. It was found that TPA can induce oxidative activation of cells, as measured by generation of H202 in response to TPA treatment in vitro (cultured HeLa cells (Frenkel and Chrzan, 1987a)) as well as in vivo (keratinocytes and epidermal cells of SENCAR mice (Perchelet et al., 1988; Robertson et al., 1990)) (Table 3). Moreover, cells exposed to TPA in vitro and in vivo also contained the oxidized bases TG, H M U and 8-hydroxyl(oxo)guanine (8-OHG) in their DNA (Frenkel and Chrzan, 1987b; Bhimani and Frenkel, 1991; Wei and Frenkel, 199 la), the structures of which are shown in Fig. 2. In vitro and in vivo formation of oxidized DNA bases was diminished by pretreatment with known antitumor promoters, such as sarcophytol A and (-).epigallocatechin gallate (EGCG) (Yoshizawa et al., 1987; Fujiki et al., 1989; Bhimani and Frenkel, 1991; Wei and Frenkel, 1991b, 1992a; Zhong et al., 1991), which were shown to also inhibit production of H202 by those cells (Fig. 3). Treatment of mouse skin with TPA causes induction and proteolytic splitting of xanthine dehydrogenase leading to the formation of xanthine oxidase (Table 3), an enzyme that mediates

0

0

...OH

I

f

R'

®

R'

R'= H; cis-TG R' = dR; ClLS-dTG

(~

O HN,,~,,,CHaOH

I

R' R' = H; HMU

R' = dR; HMdU O

O

H,N~----~I...~N

H

HaI~N..~'~.N/0

I

I

R'

R'

(-OH; 8-hydroxyl)

(=O; 8-oxo) R ' = H ; 8 - OHG R' = dR; 8 - OHdG

Where dR is 2'-deoxyrlbose

FIG. 2. Structures of selected oxidized DNA base derivatives. When R' = H, oxidized bases are thymine glycol (TG) (5,6-dihydro-5,6-dihydroxythymine); 5-hydroxymethyl uracil (HMU); and 8-hydroxyl(oxo)guanine (8-OHG). When R' = dR (2'-deoxyribose), oxidized nucleosides are thymidine glycol (dTG) (5,6-dihydro-5,6-dihydroxythymidine); 5-hydroxymethyl-2'deoxyuridine (HMdU); and 8-hydroxyl(oxo)-2'-deoxyguanosine (8-OHdG).

Carcinogen-mediated oxidant formation and oxidative DNA damage

135

OH H~O,~

....

OH

y v-.o o. OH O~p_OH (-)Eplgellocatechln Gellate (EGCG)

HO~"~O

xOH

Temoxlfen

oi.10

HOA~.~

Caffelc Acid Phenethyl Ester (CAPE)

N:'I: ' : Oltipraz

ql-~

Sarcophytol

A

CI'I2=CHCH2

CH3

Diallyl Sulfide

Curcumln FIG. 3. Structures of selected chemopreventive agents. ( - ) . Epigallocatechin gallate (EGCG); tamoxifen; caffeic acid phenethyl ester (CAPE); sarcophytol A; oltipraz; diallyl sulfide; and curcumin.

production of .O~- and H:O 2 (Reiners et al., 1987). At the same time, TPA suppresses activities of the antioxidant enzymes SOD and catalase (Solanki et al., 1981; Reiners et al., 1990), which in effect enhances the oxidative stress that develops in epidermal cells. Basal keratinocytes, which are presumed to be target epidermal cells for tumor promoters, were found to respond to TPA with oxidant formation (as detected by chemiluminescence), which was generated via protein kinase C (Fischer and Adams, 1985). ROS formation was decreased by the inhibitors of arachidonic acid metabolism as well as lipoxygenase. Metabolism of arachidonic acid causes formation of various hydroperoxides that can release oxidants. Those findings implicate release of arachidonate and its subsequent metabolism to prostaglandins as prominent responses to TPA (Fischer et al., 1985). Recently, the ability to form prostaglandin E2 was shown to distinguish TPA from its non tumor promoting isomer ~t-TPA (Fischer et al., 1991).

3. CARCINOGEN MEDIATED ROS FORMATION AND UTILIZATION 3.1. R O S PRODUCED BY IONIZING AND U.V. RADIATION

As mentioned in the Introduction, ionizing radiation generates ROS by the radiolysis of water, with .OHs being the predominant species. Under aerobic conditions, .O~- and H:O~ are also formed. Water radiolysis products and the damage they induce have been reviewed elsewhere (Scholes, 1983; Hutchinson, 1985; T6oule, 1987). Although u.v. radiation is best known for its formation of cyclobutane and non-cyclobutane pyrimidine dimers, at actinic wavelengths it also induces H202 production and consequent thymine glycol formation in cellular DNA

136

K. FRENKEL

(Leadon, 1987). When actinic irradiation occurs in the presence of metal ions, for example copper, DNA can be extensively damaged (Rossman, 1989; Rossman et al., 1989). 3.2. ROS INDUCEDBY CHEMICALCARCINOGENS Many chemical carcinogens require metabolism to electrophilic intermediates before they can bind to DNA and form base adducts (Miller, 1970; Miller and Miller, 1981). Polycyclic aromatic hydrocarbons (PAHs) are examples of this type of carcinogen. To excrete PAHs, which do not possess any reactive groups, an organism enzymatically hydroxylates them in an attempt to form soluble conjugates (Capdevila et al., 1980; White and Coon, 1980; Conney, 1982; Cavalieri and Rogan, 1985a,b). Unfortunately, during this detoxification process, more potent carcinogenic metabolites are also formed (Gelboin, 1980). It has been known for some time that certain antioxidants suppress PAH-mediated tumor formation (Shamberger et al., 1973; Wattenberg, 1980; McCormick et al., 1984; Sparnins et al., 1986; Hayatsu et al., 1988; Verma et al., 1988), but do so without reduction of the number of PAH-DNA base adducts (Dipple et al., 1984). Since adducts are thought to be a necessary starting point in chemically-induced cancer, it appears then that those antioxidants do not act during the initiation stage, but probably act during tumor promotion and/or progression. The metabolism of PAHs has been the subject of a detailed review (Conney, 1982), therefore, we will discuss only those aspects that are most relevant to ROS formation. 3.2.1. R O S Formation due to Metabolism o f Carcinogens by M i x e d Function Oxidase The primary PAH metabolizing enzyme system is a mixed function oxidase that consists of several enzymes which are present in microsomes and include aromatic hydrocarbon hydroxylase, cytochrome P450 oxidase, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase (Conney, 1982; Cavalieri and Rogan, 1985a,b; Trush and Kensler, 1991b). A variety of metabolites are formed during two electron oxidation processes, which include mono- and multi-hydroxylated products, dihydrodiols, epoxides, diol epoxides and tetrols (Gelboin, 1980). In many cases quinones are formed as well. Recently, it was shown that the cytochrome P450 system can also metabolize PAHs by one electron oxidation, which leads to the formation of a quite stable carbocation, providing that those PAHs possess a relatively low ionization potential (<7.35V) (Cavalieri and Rogan, 1990). During metabolism of PAHs (benzo(a)pyrene (B(a)P), 7,12-dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene (3MC)) by rat hepatic microsomes, substantial amounts of .02 and catalase-inhibitable H202 are generated (Frenkel et al., 1988; Frenkel, 1992). B(a)P also can be oxidized to dihydrodiols by hepatic microsomes in the presence of externally added H202 (Renneberg et al., 1981). The less carcinogenic PAHs, benzo(e)pyrene (B(e)P) and anthracene, also induce ROS formation by rat liver microsomes but in lower amounts (Frenkel, 1992). Pyrene, a non-carcinogenic parent of B(a)P and B(e)P, was totally inactive. DNA exposed to B(a)P-treated microsomes contained the oxidized thymines TG and HMU, the formation of which was suppressed by catalase (Frenkel et al., 1988). Actually, catalase decreased HMU formation by B(a)P treated microsomes below the levels present in the control microsomes in the absence of PAH. These results suggest that PAHs utilize the normal ability of microsomes to produce oxidants and enhance their generation. In addition, ROS can be formed during redox cycling of PAH quinones, as was shown to be the case for B(a)P quinones in the presence o f N A D H dehydrogenase (Lesko and Lorentzen, 1985). One of the pathways that can generate quinones is a cytochrome P450-mediated one electron oxidation of PAHs (such as B(a)P) with formation of a carbocation by the abstraction of one electron by the Fe of cytochrome P450 (Cavalieri and Rogan, 1985a,b). The reduced cytochrome P450 oxygen complex binds to the site of the positive charge of the carbocation and leaves oxygen bound to that site. After hydrogen release, an oxy-radical is formed, from which quinones are generated. Overall, three different quinones were found to be formed from B(a)P. Those and other quinones can undergo redox cycling, in which cytochrome P450 reductase mediates reduction of quinone to semiquinone (Bachur et al., 1978). The sequence of events is as follows. NADPH reduces the flavoprotein of the reductase, which in turn reduces quinone to

Carcinogen-mediated oxidant formation and oxidative DNA damage

137

semiquinone as its flavoprotein is oxidized back and ready to be reduced by another NADPH. In the presence of molecular oxygen, semiquinone reduces 02 to .O~- and becomes oxidized to quinone, which is ready to be reduced again. This cycling can occur as long as NADPH is present and it is a source of substantial amounts of .O~- and its dismutation product H202. 3.2.2. R O S Utilization by Peroxidatic Oxidation of Carcinogens Another pathway, which leads to the formation of ultimate carcinogenic metabolites, is hydroperoxide-dependent oxidation of carcinogens in reactions that are catalyzed by various peroxidases. One of the most prominent peroxidases is the myeloperoxidase of PMNs. MPO was shown to mediate preferential formation of anti diolepoxides of B(a)P (Mallet et al., 1991), the ultimate carcinogenic metabolite of B(a)P. A similar preference for anti diolepoxide formation was shown to occur when prostaglandin H synthase was utilized (Marnett and Reed, 1979; Dix and Marnett, 1983; Marnett, 1987). In this case peroxyl radicals are thought to be the intermediates. Peroxidases are present in many extrahepatic tissues, where their ability to oxidize xenobiotics and to form carcinogenic byproducts becomes a major concern (Marnett, 1987; O'Brien, 1988; Trush and Kensler, 1991a,b). In addition to PAHs, many aromatic amines and, after reduction, also nitroaromatic derivatives are oxygenated by various peroxidases to their ultimate carcinogenic metabolites (Floyd and Soong, 1977; Corbett and Corbett, 1988; O'Brien, 1988; Washburn and DiGiulio, 1988; Ritter and Malejka Giganti, 1989; Malejka Giganti et al., 1991). Thus, it appears that ROS-mediated processes can significantly contribute to the formation of tumor initiating agents that have the capacity to bind to DNA. Hence, the presence of a first stage or complete tumor promoter applied prior to carcinogen exposure (as indicated in Fig. 1B) may mediate H202 production, which can subsequently oxidize the initiator in the peroxidase-catalyzed reaction to an ultimate carcinogenic metabolite(s). That this can occur was shown when TPA treatment enhanced formation of B(a)P diol epoxide and its binding to cellular DNA in vivo (Ji and Marnett, 1991). Although complete carcinogens possess both tumor initiating and tumor promoting activities, we do not know what constitutes the tumor promoting effects of these carcinogens. We postulated that, similar to tumor promoters, complete carcinogens also induce ROS formation (Frenkel et al., 1988). These ROS could cause oxidation of DNA bases concomitant with the formation of carcinogen DNA base adducts. The concurrent presence of base adducts (representative of initiation) and oxidized bases in DNA (representative of promotion) could overwhelm the normal cellular repair processes much faster than when only a few initiating adducts are formed, followed by split dose promoter exposure over long periods of time. Preliminary data show that indeed this may be correct, since multiple DMBA treatments of mouse skin induced the in vivo formation of H202 as well as inflammatory responses (unpublished data). Moreover, at least three types of oxidized bases (TG, H M U and 8-OHG) (Fig. 2) were present in the epidermal DNA at much higher than background levels, in addition to the presence of well-known DMBA adducts. 3.3. ROS FORMATIONMEDIATEDBY CARCINOGENSTHATDO NOT BIND TO DNA The majority of carcinogens either bind to DNA directly or after metabolic activation. However, there are other agents that do not form DNA adducts yet have carcinogenic properties. One group of these agents is peroxisome proliferaters (Reddy and Lalwani, 1983; Fahl et al., 1984; Reddy and Rao, 1987). The mode of action of peroxisome proliferaters appears to depend on a 20-30 fold induction of enzymes, such as palmitoyl CoA oxidase (Table 3), which catalyze formation of H202 that is normally used in the metabolism of fatty acids (Reddy and Rao, 1987; Srinivasan and Glauert, 1990). Although the catalase levels are also enhanced, it is only by about 2 fold. Obviously, this amount of catalase cannot detoxify all of the generated H20:, since increased levels of H202 can be measured as well as the elevated presence of 8-OHG in the liver DNA. Many chemotherapeutic agents and quinone antibiotics (such as adriamycin, doxorubicin, daunorubicin and others) undergo quinone-semiquinone cycling (as described in Section 3.2.1), which leads to the formation and utilization of .O~- and H20: (as described in Section 3.2.2) (Bachur et al., 1978; Cross et al., 1987; Vuillaume, 1987). Even hormones, such as estradiol and

138

K. FRENKEL

the notorious diethylstilbestrol (DES), were shown to generate ROS during their metabolism (Table 3) (Epe et al., 1986; Roy and Liehr, 1989; Liehr and Roy, 1990; McCormick et al., 1991; Roy et al., 1991). The formation of ROS in target tissues (kidneys in the case of DES) and the oxidative damage they cause are credited for the carcinogenic activity of these agents. Redox cycling can continue until all of the NAD(P)H is utilized, which leads to depletion of cellular reductants and induces severe oxidative stress. 3.4. ROS INDUCTIONBY METAL CARCINOGENS

In addition to organic carcinogens, there are those of inorganic origin that include a number of metal derivatives (Sunderman, 1984). Although some of these metals bind to DNA others do not. Hence, their mechanism(s) of carcinogenicity must include indirect effects. Rapidly accumulating new evidence strongly suggests that these carcinogens also may act to some degree through the oxidative mechanisms (Kasprzak, 1991; Klein et al., 1991; Standeven and Wetterhahn, 1991). Chromium is readily taken up by cells as hexavalent chromate via passive anion transport channels, where it is eventually reduced to Cr(III). However, depending on the cellular reductants, Cr of the intermediate valencies also can be formed. In particular, formation of Cr(V) during reaction of Cr(VI) with microsomes, mitochondria, NADPH and ascorbate (among others) and its reactivity alone and in complex with GSH, are all related to ROS production. That this is the case was shown by inhibition of Cr(VI)- and Cr(V)-GSH-induced DNA damage by the .OH scavengers dimethyl sulfoxide, formate and benzoate, as well as catalase, which degrades H202 to water. Ni derivatives were shown to induce hepatotoxicity through formation of lipid hydroperoxides (Coogan et al., 1989), which was suppressed by benzoate, an .OH scavenger (Athar et al., 1987). Even more convincing evidence of Ni-mediated generation of ROS is the formation of many oxidized bases in DNA of cultured human cells exposed to Ni (Nackerdien et al., 1991). Other carcinogenic metal salts were also shown to cause strand breaks in cellular DNA, which could be prevented by catalase and by ROS scavengers (Snyder, 1988). These include Cd(II), Cr(VI), Hg(II) and methylmercury (Ochi et al., 1983; Snyder, 1988; Costa et al., 1992). Although Fe and Cu salts are usually not carcinogenic, some derivatives such as an Fe nitrilotriacetate ligand are carcinogenic to animals. Both Fe and Cu are mutagenic in some assays, which can be mitigated by catalase and -OH scavengers (Loeb et al., 1988; McBride et al., 1991). These results point to H202 and .OHs as being intermediates responsible for the mutagenicity of these two transition metal ions. Cu(II) can also form SOD mimetics when complexed with the appropriate ligand (i.e. (3,5-diisopropyl salicylate)2, better known as CuDIPS (Kensler et al., 1983; Egner and Kensler, 1985; Reiners and Colby, 1988)), which dismutates -Of to H2Ov In chameleon fashion, Cu(II) also mimics another enzyme, myeloperoxidase, in that it mediates oxidation of C1by H202, which results in formation of hypochlorite (Frenkel et al., 1986a). Hypochlorite is a powerful oxidant, which in addition to formation of stable chloramines (Thomas et al., 1982; Weiss et al., 1983) also can form chloramines of nucleic acids' bases by interacting with their amino groups (Bernofsky et al., 1990). These base chloramines readily form crosslinks with proteins and nucleic acids. It is not as yet known whether hypochlorite can cause an oxidative deamination of the adenine, guanine and cytosine moieties that would result in hypoxanthine, xanthine and thymine, respectively. Oxidative deamination occuring in DNA is known to be mutagenic (Singer and Grunberger, 1983). It also could provide substrates (hypoxanthine and xanthine) for xanthine oxidase, an enzyme that mediates formation of H202 (Freeman and Crapo, 1982).

4. OTHER SOURCES OF ROS 4.1. ENDOGENOUSSOURCES ROS are generated during blood reperfusion following an ischemic episode (McCord, 1985, 1987; Cross et al., 1987). During hypoxia induced by ischemia, there is a breakdown of ATP leading to accumulation of hypoxanthine, a substrate for the prooxidant enzyme xanthine oxidase that generates both .Of and H202 during the reoxygenation period. Endothelial cells themselves also can produce "02 and H202 (Sundqvist, 1991). Peroxisomes are rich sources of H202 because they

Carcinogen-mediated oxidant formation and oxidative DNA damage

139

contain an array of oxidases (Table 1) at high concentrations (Freeman and Crapo, 1982). In addition to the fatty acyl CoA oxidases, peroxisomes also include urate oxidase, D-amino acid oxidase and L-~-hydroxyacid oxidase. Cytochromes P450 and bs, present in nuclear and microsomal membranes, are ready sources of reduced oxygen species, particularly in the presence of the reductants NADPH and NADH, respectively. Hence, cells containing high levels of those cytochromes (Table 1), such as Clara cells that are present in the small airways of the lung, may be more susceptible to ROS production. The plasma membranes constitute a physical and chemical barrier that is meant to prevent the entry of damaging agents be they chemicals or radical species. However, they are easily permeated by H202, which is neutral and can cross those membranes almost as easily as water (Freeman and Crapo, 1982). 4.2. EXOGENOUSSOURCES An important source of exogenous ROS is cigarette smoke (Table 3), with the gas phase providing an oxidizing and the tar a reducing milieu (Pryor et al., 1983b, 1984; Cross et al., 1987). The gas phase rapidly inactivates the atl-proteinase inhibitor, which allows pulmonary elastase to act unchecked. Cigarette tar causes oxidative damage to DNA, probably through the stable redox cycling semiquinone. Since tar also contains transition metal ions, -OHs are readily formed, as detected by spin trapping. A number of other external agents also can mediate ROS formation (Table 3). These include ozone, which interacts with mucous membranes and forms reactive aldehydes and H202 (Pryor et al., 1983a; Borek et al., 1987; Pryor and Church, 1991) and pesticides such as paraquat (Cerutti, 1985; Vuillaume, 1987), which produce .O~- and its dismutation product H202.

5. OXIDATIVE DNA DAMAGE IN VITRO 5.1. DAMAGECAUSEDBY IONIZINGRADIATION The effects of ionizing radiation on DNA have been studied quite extensively and have been frequently reviewed (Scholes, 1983; Hutchinson, 1985; T6oule, 1987). Here, I will outline only the formation of major base derivatives produced in DNA exposed to radiation in solution or in tissue culture. It has been thought that the thymine moiety in DNA is the most susceptible to the modifying effects of ionizing radiation (Cadet and T6oule, 1978; Scholes, 1983) and, in fact, a large number of thymine oxidation products have been isolated and chemically characterized. Several hydroperoxides are also formed in response to the indirect effects of ionizing radiation (i.e. X- and ),-radiation), which generates .OHs by radiolysis of water (T6oule and Cadet, 1978; Scholes, 1983; Hutchinson, 1985; Simic and Jovanovic, 1986). 5.1.1. Thymine Moiety: Formation of Thymine Glycol (TG) and 5-Hydroxymethyl Uracil (HMU) (Fig. 4.4) The predominant thymine hydroperoxide species are cis-5(6)hydroxy-6(5)hydroperoxy-5,6-dihydrothymine. Although relatively stable, these hydroperoxides gradually decompose to the more stable cis-vicinal dihydrodiols, which are commonly referred to as cis-TG and their 2'-deoxynucleosides cis-thymidine glycols (dTG) (Cadet and T6oule, 1975a,b). There are two cis dTG diastereoisomers ((+) and ( - ) ) , which are in equilibrium with the trans epimers. This type of oxidation causes significantly altered conformation of the TG moiety by altering the planarity of thymine (Wang et al., 1979). Another hydroperoxide, 5-hydroperoxymethyl-2'-deoxyuridine(HPMdU), is derived from the methyl group of the thymine moiety (Simic and Jovanovic, 1986). HPMdU is very stable in water (Frenkel and Tofigh, 1989). However, in the presence of transition metal ions and their chelates (Tofigh and Frenkel, 1989), metalloproteins (Frenkel and Tofigh, 1989) and of horseradish or GSH peroxidases (unpublished data), HPMdU is decomposed to two major products, which are 5-hydroxymethyl-2'-deoxyuridine (HMdU) and/or 5-formyl-2'-deoxyuridine (FdU). These derivatives retain the planarity of thymidine but their methyl group is oxidized to alcohol (HMdU) or aldehyde (FdU). Both HMdU and FdU are quite stable in the presence of transition metal ions

140

K. FRENKEL

A.

--ok.J HPMz)U

HMDU

HN'~w CH3

o ,)J

+.

•

FDU

O ~.CH 3

o---i:--o.

DT

o

.N"U"FS '

~

P

cls-oTG

,, cts-S(6)-DTOOH

WHERE: R'= DR (2'-DEOXYRIBOSE) O

B.

OH ~ N H cH3 o

0

HN~,~SH3

H N ' ~ CH3

-

H TG

-

J

HMH

o+L...c.o H

FPU

~'~,u,r,,C.O N'- FORMYI_-N-UREA

FIG. 4. Structures of some 7-radiation mediated products of thymidine oxidation. (A) dT (thymidine); HPMdU (5-hydroperoxymethyl-2'-deoxyuridine); HMdU (5-hydroxymethyl-2'deoxyuridine); FdU (5-formyl-2'-deoxyuridine); cis-5(6)-dTOOH (cis-5(6)-hydroxy-6(5)-hydroperoxy-5,6-dihydrothymidine); and cis-dTG (cis-thymidine glycol; 5,6-dihydroxy5,6-dihydrothymidine); where R'= dR (2'-deoxyribose). (B) TG (thymine glycol; 5,6-dihydroxy-5,6-dihydrothymine); FPU (N'-formyl-N-pyruvylurea); HMH (5-hydroxyl-5-methylhydantoin); and N'-formyl-N-urea.

or peroxidases. The dTG moiety, however, is easily altered in the oxidizing milieu induced by ionizing radiation or by H202 in the presence of Fe(II) chelates (Frenk¢l et al., 1986b). The gradual oxidative decomposition of dTG (Fig. 4B) leads to opening of a TG ring with formation of N'-formyl N-pyruvylurea (FPU), which can either become a five membered ring of 5-hydroxyl-5-methylhydantoin (HMH) or be degraded further to the N'-formyl-N-urea, N'-formyl or N-urea residues (Cadet and T6oul¢, 1975b; T6oule et al., 1977). Hence, there are a large number of degradation products derived from the dTG moiety in DNA. Assays that measure only intact dTG may therefore underestimate the actual formation of dTG. Often it appears that more dTG is present under conditions that inhibit .OH formation (unpublished data), but this is only because under those conditions the TG ring is more protected from degradation.

Carcinogen-mediated oxidant formation and oxidative DNA damage O HN"

I

141

H

~

C

II

H,aN~"~N~'~N Hz

\H

FapyG

H

0

H2

FapyA FIG. 5.

Structures of FapyG and FapyA. FapyG (2,6-diamino-4-hydroxy-5-formamidopyrimidine; and FapyA (5-formamido-4,6-diaminopyrimidine).

5.1.2. Other DNA Bases: Formation of 8-Hydroxyl(oxo)guanine (8-OHG) In addition to oxidized thymines, the other DNA bases are also subject to the oxidizing effects of ionizing radiation (Dizdaroglu, 1985; Teebor et al., 1988). For example, oxidation of cytosine results in formation of a number of products by oxidative deamination that are analogous to those of thymine. Purines can be oxidized at many positions, but those modified at the C-8 of the imidazole ring have been studied more extensively than the others. One of the most frequently studied oxidized derivatives is that with a hydroxyl (oxo) at the C-8 of guanine (Fig. 2), due to its easy electrochemical detection as the nucleoside 8-hydroxyl-2'-deoxyguanosine (8-OHdG) (Floyd et al., 1986), which is also referred to as 8-oxo-dG (Cho et al., 1990; Floyd, 1990; Tchou et al., 1991). Purines substituted with -OH (oxo) at the C-8 can undergo an imidazole ring opening (Dizdaroglu, 1991) that is similar to that of the purines substituted at the N-7 with an alkyl group. Upon ring opening, appropriately substituted pyrimidine derivatives are formed. These derivatives are often called FapyG or FapyA (Fig. 5), where the Fapy stands for 2,6-diamino-4-hydroxy-5-formamidopyrimidine or 5-formamido-4,6-diaminopyrimidine, respectively. Those open ring derivatives appear to be present in irradiated DNA in addition to the oxidized purines (Dizdaroglu, 1991). 5.1.3. Analysis of DNA: Quantitation of Oxidized DNA Bases Over the years, the most frequently analyzed oxidized base derivatives in DNA in solution or in ceils exposed to ionizing radiation have been dTG, H M d U and 8-OHdG (Cerutti, 1976; T6oule et al., 1977; Frenkel et al., 1981, 1985, 1986b; Teebor et al., 1984, 1987; Kasai et al., 1986; Floyd, 1990). Since the methods used for DNA analysis differ among laboratories, it is difficult to evaluate the relative amounts of those bases in DNA. With the development of new methods that do not require prelabeling of DNA (Dizdaroglu, 1991; Frenkel et al., 1991b), the question of the relative abundance of those oxidized base derivatives should be answered in the near future. Usually, it is anticipated that the damage induced in DNA irradiated in solution would be much higher than that induced in cellular DNA, whether in cultured ceils or in vivo. However, using the G value (amount of H M d U formed/100 eV of radiation), obtained for H M d U formed in DNA solutions through the indirect effects of ionizing radiation (due to the radiolysis of water), the predicted levels of H M d U in the DNA of HeLa cells were much lower than the actual measured levels (Frenkel et aL, 1985). These results were probably due to cumulative indirect and direct effects of ionizing radiation that cause cellular DNA damage. The involvement of direct effects in DNA damage becomes more apparent when one considers the concentration of DNA in the nucleus, which is very high (Frenkel et al., 1985). The direct effects may actually predominate because, at that high concentration and by being a part of chromatin, DNA is almost like a solid matrix in which the indirect effects of radiation are minimal (Cadet and Berger, 1985). The G value for the J'PT 53/I--J

142

K. FRENKEL

formation of dTG in DNA irradiated in solution (G = 0.002) was comparable to that of H M d U with about equal amounts of ( + ) and ( - ) cis dTG present in the irradiated DNA (Teebor et al., 1987). 8-OHdG was also found to be present in D N A of y-irradiated HeLa cells (Kasai et al., 1986). Using GC-MS, Dizdaroglu (1985) identified many derivatives formed in irradiated DNA by analyzing oxidized D N A bases released by formic acid hydrolysis and acylated with the trimethylsilyl groups. Using that method, most of the derivatives of all four D N A bases were identified and quantified. Conspicuous in its absence was HMU. It seems possible that the conditions of the formic acid hydrolysis of DNA were not appropriate for the release of free HMU, whereas other investigators, who found H M U in DNA, hydrolyzed DNA enzymatically to nucleosides (Frenkel et al., 1985, 1986b), which is a much gentler technique. Just recently, Djuric et al. (1991b) using GC-MS showed that the acid hydrolysis method detects only about half of the H M U residues as those obtained by enzymatic hydrolysis. When the DNA in chromatin was irradiated, the yields of oxidized bases were lower than those present in DNA irradiated in solution, which points to histones of chromatin as providing an effective protective barrier (Gajewski et al., 1991; Dizdaroglu 1991). 5.2. DAMAGECAUSED BY TUMOR PROMOTERS In recent years, a substantial amount of work has been carried out in several laboratories based on the premise that if tumor promoters induce ROS formation in cells, those ROS might cause heritable genetic damage (Table 4). The first reports, showing that ROS produced by TPA-activated PMNs mediate formation of DNA strand breaks in its own DNA as well as in the co-incubated mouse erythroleukemia cells, were published by Birnboim (1982, 1983). These studies were followed by the work of Dutton and Bowden (1985), who found that ROS produced by TPA-stimulated macrophages also caused strand breaks in co-incubated epidermal cells. TABLE4. H2 O2 and Oxidative D N A Damage Formation In Vitro Agent v-Radiation u.v.-radiation PMNs/TPA, mezerein or RPA PMNs/TPA PMNs/TPA Macrophages/TPA TPA TPA DMBA and other PAHs B(a)P B(a)P 4-Nitroquinoline N-oxide Asbestos Cr(VI), Cr(V)GSH Ni acetate Betel nut extract Ozone Cigarette smoke DES quinone Cd(II), Hg(II), methyl mercury

H202

HMU, TG or 8-OHG

+ + + + + + + + + + nd + nd + nd + + + + nd

+ + + + +t +t + nd:~ nd + + + + + + + +t +t + +t

Target DNA; HeLa cells Mammary epithelial cells (MEC) DNA PMNs, HeLa cells, MRC5* MEL cells Epidermal cells HeLa cells Keratinocytes Rat liver microsomes Rat liver microsomes MEC Ehrlich ascites cells DNA Human fibroblasts; DNA DNA in chromatin DNA DNA DNA Kidney cortex microsomes: DNA Human fibroblasts; CliO

References [a] [b] [c] [d] [e] [f] [d, g] [h] [i, j] [i] [k] [1] [m] [n] [o] [p] [q] [r] [s] [t]

*Unpublished data (H. Wei, D. Corvese, A. Weitberg and K. Frenkel). tStrand breaks were measured, not oxidized DNA bases. :~nd: Not determined. [a] Teebor et al., 1984; Frenkel et al., 1985; [b] Leadon, 1987; [c] Frenkel and Chrzan, 1987a; [d] Frenkel and Chrzan, 1987b; [e] Birnboim, 1983; [f] Dutton and Bowden, 1985; [g] Bhimani and Frenkel, 1991; Frenkel and Gleichauf, 1991; [h] Robertson et al., 1990; [i] Frenkel et al., 1988; [j] Frenkel, 1989a, 1992; [k] Ide et al., 1983; [1] Kohda et al., 1986; [m] Kasai and Nishimura, 1984b; In] Snyder, 1988; Aiyar et al., 1989; Kortenkamp et al., 1990; Standeven and Wetterhahn, 1991; [o] Nackerdien et al., 1991; [p] Nair et al., 1987; [q] Borek et al., 1987; Pryor, 1987; [r] Cross et al., 1987; [s] Epe et al., 1986; Roy et al., 1991; [t] Ochi et al., 1983; Snyder, 1988; Costa et al., 1992.

Carcinogen-mediated oxidant formation and oxidative DNA damage

143

TABLE5. H202 and Oxidative DNA Damage Formation in vivo

Agent 1. Tumor promoters --TPA, mezerein, RPA 2. Inorganic carcinogens --KBrO3 --Ni acetate --Fe-nitrilotriacetate 3. Organic carcinogens --DMBA --B(a)P --Nitroalkanes --Cyclopentanone oxime --DES --Peroxisome proliferators --Diethylnitrosamine --2-Acetylaminofluorene 4. Physical carcinogens --y-Radiation

H20~

HMU, TG and/or or 8-OHG

Site

References

+

+

mouse skin

[a]

nd nd nd

+ +* +

rat kidney rat kidney rat kidney

[b] [c] [d]

+ nd nd nd + + nd nd

+* nd* + + + + -I+

mouse skin mouse skin rat liver rat liver hamster kidney rat liver rat liver rat liver

nd

+

rat liver

[e] [f] [g, h] [h] [i] [j, k] [k] [k] [k]

nd: Not determined. *Inflammatory response. [a] Wei and Frenkel, 1991a, 1992c; [b] Kasai et al., 1987; [c] Kasprzak et al., 1991; [d] Umemura et al., 1990; [e] Wei and Frenkel, 1992d; [f] Albert et al., 1991; [g] Fiala et al., 1989; [h] Conaway et al., 1991; [i] Roy et al., 1991; [j] Kasai et al., 1987; [k] Srinivasan and Glauert, 1990. 5.2.1. Formation o f TG, H M U , 5-Formyl Uracil and 8 - O H G in D N A In the meantime, initial reports were published showing formation of oxidized DNA base derivatives (HMdU and dTG) by ROS generated by TPA-mediated activation of PMNs or macrophages (Troll et al, 1984; Lewis and Adams, 1985; Frenkel et al., 1986b). This effect was also shown for other tumor promoters (Frenkel and Chrzan, 1987a) (Table 4). HMdU was found to be formed in a dose-dependent manner in the DNA of HeLa cells coincubated with TPA-stimulated PMNs (Frenkel and Chrzan, 1987b). That work also led to the discovery that a tumor promoter can induce oxidative activation of cells even in the absence of PMNs, which was deduced from the TPA dose-dependent formation of HMdU in HeLa cell DNA (Frenkel and Chrzan, 1987b, Frenkel, 1989a). Because of this finding, an effort was made to actually measure the ROS produced by the TPA-induced process(es) in HeLa cells. As it turned out, H202 was the predominant ROS produced (Frenkel and Gleichauf, 1991). This formation of H202 could be prevented by preincubation with EGCG (Bhimani and Frenkel, 1991) (Table 5, Fig. 3), a polyphenolic tannin isolated from green tea leaves, which is known to exhibit anti-tumor promoting activity in vivo (Yoshizawa et al., 1987; Suganuma et al., 1992). With higher doses of EGCG, we observed a decrease in H202 below control levels in HeLa cells that were not treated with TPA. These results suggest that TPA enhances the output of the preexisting H202-generating proeess(es). In addition to H:O2, the formation of HMdU, dTG and 8-OHdG was also decreased in HeLa cell DNA by EGCG (unpublished data) (Table 5). Incubation of DNA in solution with H20: in the presence of Fe(II)/EDTA caused a timedependent formation of dTG, HMdU, FdU and 8-OHdG, with the amounts of the last two derivatives being comparable and the lowest of the four (Frenkel et al., 1991b). This was the first demonstration of FdU formation in DNA exposed to the oxidative stress. Concurrently, Kasai and Nishimura (1984a,b) found that DNA exposed to H202 in the presence of transition metal ions (Fe or Cu) or asbestos contained 8-OHdG (Table 4). These findings were followed in quick succession by a demonstration that 8-OHdG is present in the DNA of PMNs, which were stimulated by TPA (Floyd et al., 1986) and in isolated DNA that was exposed to .OHs or singlet oxygen (Floyd et al., 1988; Schneider et al., 1990). Dizdaroglu's laboratory analyzed DNA oxidized by a variety of .OH-generating systems, such as the Fe-dependent effects of .O~- or H20: (Aruoma et al., 1989a,b) and ROS generated by

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TPA-stimulated PMNs (Jackson et al., 1989). Recently, they also analyzed oxidized bases in DNA that was treated in chromatin with H202 in the presence of Fe or Cu (Dizdaroglu et al., 1991) (Table 4). This method of analysis utilizes selectivity and sensitivity of GC coupled to MS. The only real problem with this method is that it requires very expensive equipment, which is only available to a few laboratories. There is another method that can be applied by most laboratories without the need for the specialized equipment. Utilization of this method (3H-postlabeling) results in a greatly improved sensitivity of detection, down to pmole amounts of each of the analyzed base derivatives. These derivatives can also be simultaneously determined (Frenkel et al., 1991b) and applied to the analysis of DNA isolated from in vivo-treated animals (Wei and Frenkel, 1991a) and from human white blood cells (Bhimani et al., 1992). 5.3. DAMAGE CAUSED BY CHEMICAL CARCINOGENS 5.3.1. Oxidative D N A Modification by Benzo(a)pyrene [B(a)P] It has been realized for some time that antioxidants suppress the development of tumors induced by PAHs (Wattenberg, 1980; Dipple et al., 1984; Verma et al., 1988), at least one of which [B(a)P] causes oxidative DNA damage in cells, which exceeds the level of adduct formation by about 20 fold Ode et al., 1983). More recently, the types of oxidative base damage induced by PAHs were identified. Leadon (1987; Leadon et al., 1988) using monoclonal antibody showed that B(a)P actually mediates formation of TG in DNA of mammary epithelial cells (Table 4) and that the arachidonic cascade seems to be involved in its formation (Tischler and Leadon, 1990). Frenkel et al. (1988) showed that treatment of microsomes (isolated from Sprague-Dawley or Fisher 344 rat liver) with B(a)P induces generation of ROS, which causes oxidation of the thymine moiety in coincubated DNA to TG and H M U (Table 4). The levels of these oxidized bases were 6- and 4.5-fold higher in those two rat strains, respectively, than in the control DNA [in the absence of B(a)P]. Catalase inhibited formation of H M U even below the levels present in control DNA coincubated with Fisher 344 microsomes. This finding points to H202 as being a ROS intermediate, which is necessary for PAH-mediated oxidative DNA damage and that B(a)P metabolism utilizes the normal cellular microsomal machinery, which is a likely source of the background oxidative damage formation. 5.3.2. Oxidative D N A Modification by Other Carcinogens In addition to B(a)P, other carcinogens also cause oxidative DNA damage in vitro (Table 4). Despite their diversity, both u.v. radiation and N-hydroxy-2-naphthylamine cause formation of TG in cellular DNA (Leadon, 1987). Ehrlich ascites cells treated with 4-nitroquinoline-N-oxide exhibit 8-OHG in their DNA (Kohda et al., 1986), which the authors thought resulted from removal of the more typical guanine adducts. However, as noted by others, another mechanism is also possible (Vuillaume, 1987). During reductive metabolism, this carcinogen can transfer an electron to molecular oxygen, which would lead to formation of .Of and H~O2. It was also noted by Trush and Kensler (1991 b) that 4-nitroquinoline-N-oxide can undergo redox cycling. It is more likely that the formation of 8-OHG in cellular DNA is mediated by HzO2, rather than being a secondary product formed from an adduct. Many other agents that are capable of generating oxidants also induce oxidative DNA damage, as measured by 8-OHdG. Those carcinogens include asbestos, which in the presence of H202 causes DNA base oxidation (Kasai and Nishimura, 1984b). In light of that finding, H202 generated by macrophages that partially phagocytize asbestos particles (futile phagocytosis) (Hei and Kushner, 1987) could be a source of the damage. The Cr(V)-GSH complex was also shown to induce 8-OHG formation in DNA (Aiyar et al., 1989), as did the extract from betel nuts (Nair et al., 1987). Another environmental source, cigarette smoke, was found to cause DNA strand breaks (Pryor et al., 1983b, 1984; Cross et al., 1987). Strand breaks are indicative of .OH-mediated damage and usually their presence is predictive of the presence of oxidized bases in DNA as well. The tar condensate from cigarette smoke consists of a large variety of agents, which include reducing equivalents that are needed for production of .Of from the molecular oxygen. It also contains transition metal ions, which reduce H202 obtained by dismutation from ,Of (Cross et aL, 1987). Formation of numerous

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oxidation products in chromatin exposed to nickel acetate was also recently shown (Nackerdien et al., 1991).

6. OXIDATIVE DNA DAMAGE IN VIVO AND ITS PREVENTION 6.1. DNA DAMAGEINDUCEDBY TUMORPROMOTERS

6.1.1. 12-0-Tetradecanoyl-phorbol- 13-acetate (TPA)-induced Formation of TG, H M U and 8-OHG Until recently, only one preliminary meeting report showed that the tumor promoter TPA is capable of inducing 8-OHG formation in mouse skin DNA (Floyd et al., 1987). In a full report just published by Wei and Frenkel (1991a), it was shown that TPA causes time- and dose-dependent formation of at least three types of oxidized bases, as measured by dTG, HMdU and 8-OHdG in epidermal DNA of TPA-treated SENCAR mice (Table 5). The use of a newly-developed 3H-postlabeling assay, utilizing HPLC separation of nucleosides and their acetylation with [3H]acetic anhydride (Frenkel et al., 1991b), also revealed differences in the rates of formation of ( + ) and ( - ) cis-dTG isomers. Formation of oxidized thymidines was maximal 8 hr after treatment with 4 /~g (6.5 nmol) TPA (29.1 and 17.3/104 nucleosides for dTG and HMdU, respectively), but there was a smaller peak of activity at 1-2 hr (22.8 and 13.0/104, respectively). 8-OHdG was formed in much smaller amounts, reached the maximum (3.2/10 s nucleosides) in 6 hr and remained elevated for an additional 18 hr. Increasing the TPA dose from 2/~g to 4 #g caused an increase in all three derivatives. However at l0 btg, only 8-OHdG levels still increased but both dTG and HMdU declined. That decline in oxidized thymidines paralleled the results of in vivo experiments, in which SENCAR mice were initiated with one dose of DMBA and promoted with TPA. In those experiments, the 10/~g dose of TPA was toxic and resulted in a decrease in the number and multiplicity of tumors in comparison to 5 #g TPA (Slaga, 1983). Finding that 8-OHdG was still enhanced at this toxic dose points to the differences in the mechanism of its formation in comparison to dTG and HMdU. All three types of oxidized bases can be formed by the action of .OHs (Frenkel, 1989a; Floyd, 1990). However, 8-OHdG was shown to be formed also by the singlet oxygen, whereas others are not (Floyd et al., 1989; Schneider et al., 1990). 6.1.2. Comparison of TPA Effects with those of Mezerein and 12-O-Retinoyl-phorbol-13-acetate (RPA) To determine whether ROS levels and the consequent oxidative DNA damage are related to in vivo effects of tumor promoters, in addition to TPA, two weaker tumor promoters (mezerein and RPA) were applied to the mouse skin. The extent of H202 production in the mouse skin and formation of oxidized thymines in cellular DNA were significantly lower when these two promoters were used than in the case of TPA (Wei and Frenkel, 1992c). In contrast, the ability to recruit PMNs to mouse skin was comparable for TPA and mezerein and although it was lower when RPA was used the difference was not statistically significant. These results point to H202 formation by both epidermal cells and infiltrating PMNs as being related to the potency of in vivo tumor promotion, while the ability to recruit PMNs is not. The differences among these three tumor promoters were already evident when human PMNs were activated by them in vitro. As measured by H202 production and formation of dTG and HMdU in co-incubated DNA in the absence or presence of autologous plasma, TPA was the most effective, followed by mezerein and RPA, which was the same order as their effectiveness in vivo (Frenkel and Chrzan, 1987a; Frenkel, 1989a). Similar conclusions were reached by Sirak et al. (1991), who analyzed PMNs isolated from peripheral blood for their ability to produce H202 24 hr following exposure of mouse skin to TPA, mezerein, 12,13-phorbol-dibutyrate or 4-O-methyl-TPA. The rank order of potency declined with TPA being the highest, followed by mezerein and then dibutyrate. 4-O-methyl-TPA, which is a very weak tumor promoter, was inactive. The authors suggested that the potency of the tumor promoters may be based on the activation of circulating leukocytes and on the stimulation of ROS formation by epidermal cells and not on the ability of PMNs to infiltrate dermis.

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K. FRENKEL TABLE6. Inhibition of Carcinogenesis, ROS and Oxidative DNA Base Damage by Chemopreventive Agents

Inhibition of HMU, TG Agent Carcinogenesis ROS or 8-OHG References Sarcophytol A + + + [a-c] EGCG + + + [d] CAPE nd + + [e] Tamoxifen + + + Ill Protease inhibitors + + + [g] Potato inhibitors 1 and 2 Chicken ovoinhibitor Bowman-Birk inhibitor Garlic and onion oils, + nd nd [h] n-propyl disulfide and alkyl methyl trisulfide Curcumin + nd nd [i] nd: Not determined. [a] Narisawa et al., 1989; [b] Frenkel et al., 1991a; [c] Frenkel, 1992; [d] Yoshizawa et al., 1987; Bhimani and Frenkel, 1991; Wei and Frenkel, 1992c; [e] Frenkel et al., 1992; If] Buckley and Goa, 1989; Troll and Lim, 1991; Wei and Frenkel, 1992c; Zhong et al., 1991; [g] Frenkel et al., 1987, 1991a; Troll et al., 1987; Weed et al., 1985; [h] Belman, 1983; Sparnins et al., 1986; Belman et al., 1989; [i] Huang et al., 1988, 1991. 6.1.3. Effect o f Anti-tumor Promoters on T P A - m e d i a t e d D N A Damage (Table 6) Cumulatively, the results presented above strongly suggest that it is the production of H202 and oxidative DNA damage that is intimately involved in tumor promotion. To prove this point, we pretreated SENCAR mice (30 min before TPA application) with the known anti-tumor promoters, EGCG (from green tea leaves) and sarcophytol A (isolated from soft marine coral (Kobayashi et al., 1979; Yoshizawa et al., 1987; Fujiki et al., 1989; Fujita et al., 1989; Narisawa et al., 1989; Suganuma et al., 1992)) and with caffeic phenethyl ester (CAPE) a potential anti-carcinogen (Grunberger et al., 1988) and tamoxifen, an anti-inflammatory anti-cancer agent (Buckley and Goa, 1989; Troll and Lim, 1991). Although the structures of these substances are so different (Fig. 3), they all protected the mouse skin from an inflammatory response to TPA, which was also reflected by lower levels of H202 and oxidized DNA bases (Frenkel et al., 1992b; Wei and Frenkel, 1991b, 1992a,c). Similar results were obtained when human PMNs were pretreated with these agents before stimulation with TPA, in that H202 production was suppressed by all of these substances (Frenkel et al., 1991a; Troll and Lim, 1991; Zhong et al., 1991). Moreover, sarcophytol A and protease inhibitors that recognize chymotrypsin caused a decrease not only in H202 but also in oxidation of bases in coincubated DNA (Table 6) (Frenkel et al., 1987, 1991a; Frenkel, 1992). Hence, both in vitro as well as in vivo experiments point to H202 and the consequent oxidative DNA base modification as being active participants in tumor promotion, since anti-tumor promoters inhibit both of these parameters as they suppress tumor formation. A number of other agents were found to act as anti-carcinogens in vivo and, at the same time, to possess anti-oxidant activities (Perchelet and Perchelet, 1989). Those agents also include diverse substances such as diallyl sulfide and other garlic and onion constituents, curcumin as well as oltipraz and their derivatives (Fig. 3) (Belman, 1983; Perchelet et al., 1986; Sparnins et al., 1986; Goldberg, 1987; Huang et al., 1988, 1991; Belman et al., 1989; Gali et al., 1991; Kensler et al., 1991). Although these chemopreventive agents may differ in their mechanisms of action, i.e. inhibition of prooxidant enzymes as opposed to induction of the detoxifying enzymes, the overall effect can be quite similar, that is suppression of carcinogenesis in vivo.

6.1.4. TPA-induced D N A Modification in Epidermis o f S E N C A R versus C 5 7 B L / 6 J Mice SENCAR mice are sensitive to TPA-mediated tumor promotion, while C57BL/6J mice are quite resistant to the regimen in which mice are treated twice/week for a prolonged period (Slaga, 1983).

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However, it is not known what predisposes these mouse strains to their particular responses. It was shown that macrophages isolated from SENCAR mice generated four times as much H202 in response to TPA stimulation as those from C57BL/6J mice (Lewis and Adams, 1986). These findings indicate that differences in the ability to form ROS may be responsible for the species differences in TPA-mediated tumor promotion. We recently completed experiments that show the quantitative differences in hyperplasia, infiltration of PMNs, H~O2 production and oxidized DNA base formation (Wei and Frenkel, 1992b). All of these parameters were significantly lower in the C57BL/6J than in the SENCAR mice. In a recent study, Kasprzak et al. (1991) showed that the 8-OHdG formation in renal DNA in response to i.p. treatment with Ni(II) acetate was barely enhanced (13%) in C57BL mice but was substantially increased (by 150%) in BALB/c mice, which were also more susceptible to lipid peroxidation induced by this Ni derivative (Rodriguez et al., 1991; Misra et al., 1991). These results also suggest that it is the ability to generate oxidants and oxidative damage (this time to the lipids, which potentially can mediate oxidation of DNA bases) that is related to the carcinogenic potential of an agent. 6.2. CARCINOGEN-MEDIATEDFORMATIONOF OXIDIZEDDNA BASES 6.2.1. By Inorganic Carcinogens Since 1987, many published reports showed that oxidative DNA damage also occurs in vivo when animals are exposed by different routes to a variety of carcinogens (Table 6). One of the first reports showed that feeding rats KBrO 3 caused formation of 8-OHG in kidneys, which are the target organ for its carcinogenicity (Kasai et al., 1987). Similarly, nickel acetate and Fe-nitrilotriacetate caused formation of 8-OHdG in the DNA of kidneys, again a target organ, but not in liver DNA, which is not a target tissue for these agents (Kasprzak et al., 1990; Umemura et al., 1990). These data point to kidneys as being especially sensitive to the effects of metal carcinogens, possibly because of the response, i.e. accumulation of metal salts and the cellular formation of ROS, and/or the inflammation caused by infiltrating phagocytes. 6.2.2. By 7,12-Dimethylbenz(a)anthracene (DMBA) and B(a)P In our quest to define the tumor promoting effects of complete carcinogens, which require metabolic activation for their carcinogenicity, we tested the hypothesis that these carcinogens (i.e. PAHs) induce oxidant production in vivo with a consequent formation of oxidized DNA bases. SENCAR mice were treated with DMBA (100 nmol) or TPA (6.5 nmol) for 5 weeks, 2x/week and then several parameters were followed for an additional 5-10 weeks. These included infiltration of PMNs (as quantitated by MPO), H~Oz production and formation of the oxidized DNA base derivatives H M d U and 8-OHdG (Wei and Frenkel, 1991a, 1992a), as well as tumors (Table 5). The preliminary results (Wei and Frenkel, 1992d) show that the inflammatory response was higher in animals treated with 10 doses of DMBA than with 10 doses of TPA and remained much higher for at least 3 weeks after the last application than that which was mediated by TPA, which declined rapidly. In contrast, HzO2 was present at higher levels after TPA treatment than after DMBA. Both types of treatment enhanced formation of HMdU and 8-OHdG, but the kinetics of their appearance and the subsequent decline were quite different between DMBA- and TPA-treated mice. Decrease in the TPA-induced 8-OHdG was gradual. In the case of DMBA, there was a rapid decline to about 30% of the level within 24 hr after the tenth application where it remained for several weeks, which was long after that induced by TPA reached the baseline. TPA-mediated formation of H M d U was the highest 24 hr after the tenth application, while that induced by DMBA was still increasing 48 hr past the tenth DMBA treatment. Tumors appeared and started to grow rapidly 2 weeks after the tenth DMBA treatment but not after TPA. The described preliminary results show that PAHs such as DMBA cause oxidant production and oxidative DNA damage in vivo at the same time that the adducts are formed between PAH metabolites and DNA bases. Recently, a study by Albert et al. (1991) showed that mouse treatment with B(a)P, another PAH, causes many biochemical changes, which differ depending on the dose of the carcinogen. The number of DNA adducts increased linearly in the epidermis with doses up to 32 #g/week but not at 64/~g/week. In contrast, a number of tumors increased sharply above the 32/~g/week dose.

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Hence, the number of tumors did not correlate with the adducts. The authors noted an enhanced inflammatory response in the dermis at all B(a)P doses and a presence of dark keratinocytes. The conclusion drawn was that the damage induced by the highest dose of B(a)P also reflected tumor promoting activity because at that dose there was significant cell killing and proliferative regeneration, which are the hallmarks of tumor promotion. Although different model systems and different PAHs were used in the last two studies, the conclusions appear to be the same. Inflammatory responses cause both ROS production and oxidative DNA base modification, while proliferative regeneration may fix the DNA damage as mutations prior to its repair. The presence of 8-OHdG in oligonucleotides was shown to lead to mispairing, whereas 8-OHdG as well as HMdU are mutagenic (Kuchino et al., 1987; Shirnam6 Mor6 et al., 1987; Boorstein and Teebor, 1988; Wood et al., 1990; Shibutani et al., 1991) Although sometimes mutagenic (Basu et al., 1989), the presence of dTG appears to be predominantly a replication block (Ide et al., 1985; Rouet and Essigman, 1985), which could represent the cytotoxic effects of tumor promoters. 6.2.3. By other Organic Carcinogens (Table 6) Recently, it was shown that not only 2-nitropropane but also a whole group of nitroalkanes, cyclopentanone oxime and other related compounds cause formation of 8-OHG in DNA as well as in RNA of rat liver, which is the site of carcinogenicity for those chemicals (Fiala et al., 1989; Conaway et al., 1991). As measured by HMdU formation, treatment of rats with diverse carcinogens (e.g. y-radiation, diethylnitrosamine, 2-acetylaminofluorene and ciprofibrate (a peroxisome proliferater)) caused oxidative DNA damage in hepatic DNA (Srinivasan and Glauert, 1990). The extent of that damage was dependent on the carcinogen dose and exposure time. That peroxisome proliferaters can induce oxidative modification of DNA bases was already indicated by the results of Kasai et al. (1989). They showed that 8-OHdG is formed in the DNA of rat liver in response to the chronic treatment, which usually occurs when such drugs are used by humans. Peroxisome proliferaters are examples of carcinogens that do not form DNA base adducts. However as mentioned in the Section 3.3, they do cause a multi-fold induction of H202-producing enzymes (Reddy and Lalwani, 1983), which leads to oxidative DNA damage (Kasai et al., 1987; Srinivasan and Glauert, 1990). Another example of carcinogens enhancing oxidative stress in vivo is DES. It used to be given as an anti-nausea drug to pregnant women but was subsequently taken off the market because the daughters of those women were found to be very prone to cancers. DES forms quinones and undergoes redox cycling both in vitro and in vivo (Liehr and Roy, 1990). Recently, this agent was shown to induce formation of 8-OHdG in the kidney DNA of male Syrian hamsters when they were chronically exposed to this drug through implants, but not in the liver (Roy et al., 1991). Syrian hamsters are very sensitive to estrogen-mediated tumor development, which occurs in kidneys but not in liver (Roy and Liehr, 1989; McCormick et al., 1991). Interestingly, more acute exposure induced increased 8-OHdG formation in both of these organs (Roy et al., 1991). This demonstrates the importance of utilizing an appropriate route and timing of exposure in order to get a meaningful animal model system. Although it was already known that the antioxidant defenses in DES-treated Syrian hamsters were decreased (Roy and Liehr, 1989), recent results show that the levels of both constitutive SOD (CuZnSOD) and inducible mitochondrial SOD (MnSOD) and of catalase are significantly lower in renal tumors than in the surrounding tissue (McCormick et al., 1991). 6.3. OXIDATIVE D N A DAMAGE IN HUMANS

It has been a long-standing assumption that dietary consumption of fat contributes to cancer development in humans. However, there were no markers of exposure that could be easily utilized to test this assumption. Recently, Djuric et al. (1991a) analyzed the DNA of white cells isolated from human blood for the presence of HMU. Those analyses showed that in women with a high risk for breast cancer a dietary change lowering fat intake can decrease oxidative DNA damage. The decline in measured HMU was highly significant (p < 0.005) in the diet intervention group as

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compared to the group that remained on a high-fat diet. It is presumed that the decrease in fat consumption is compensated by an increase in the intake of other foods such as vegetables, grains, seeds and fruit. These foods are known to lower the production of ROS and formation of oxidative DNA damage in vitro as well as the incidence of human cancer (Correa, 1981; Frenkel et aL, 1987; Troll et al., 1987). It remains to be seen whether the women with reduced oxidative damage in their white blood cell DNA will also be protected from breast cancer. This type of protection seems likely because it was noticed that alteration in dietary consumption of fat and specific fatty acids causes the reduction of tumor development in animals (Ip et al., 1985; Roebuck et al., 1985; Fisher et al., 1991; Lockniscar et al., 1991). Just recently, a brief report was published showing that DNA of female breast invasive ductal carcinomas contained nearly an order of magnitude higher levels of oxidized bases than normal DNA controls (Malins and Haimanot, 1991). Among the DNA samples from five different carcinomas, 8-OHG, 8-hydroxyladenine and FapyG (but not FapyA) were significantly increased in four specimens and only were doubled in the fifth. Oxidized pyrimidines were not measured. The distribution of the three oxidized base derivatives varied among the DNA samples. Interestingly, DNA isolated from the surgical margins was not significantly different from the control DNA with respect to the content of these oxidized bases. These results point to .OH-mediated DNA damage as being a likely contributor to the malignant properties of the tumors. Since tumor cells were shown to produce high levels of H202 (Frenkel and Gleichauf, 1991; Szatrowski and Nathan, 1991), that oxidant might potentiate the DNA damage and enhance genetic instability (Malins and Haimanot, 1991). Based on the premise that inflammatory conditions induce oxidant production and oxidative DNA damage and on the fact that many of these patients develop anti-DNA antibodies (Niwa et al., 1985; Cross et al., 1987; Blount et al., 1990; Weitzman and Gordon, 1990), we decided to look for the presence of antibodies that recognize oxidized DNA bases. The sera of people, who were tested for the presence of anti-nuclear antibodies and from healthy controls, were assayed using the enzyme-linked immunosorbent assay (ELISA) with H M d U coupled to bovine serum albumin (HMdU-BSA) as the antigen for coating the wells. We found that the sera of people with inflammatory diseases contained significantly higher (p < 0.001) levels of antibodies that bound to the HMdU-BSA-coated wells than those of the healthy controls (Kim and Frenkel, 1990) and those whose sera contained anti-nuclear antibodies but did not have a clinically active disease (K. Frenkel, J. Karkoszka, E. Kim and J.-C. Bystryn, submitted for publication). Since people, who have diseases such as systemic lupus erythematosus that are inflammatory in nature, also are known to have a higher incidence of cancer (Emerit et al., 1980; Cross et al., 1987), it may be possible to use the presence of anti-oxidized DNA antibodies as an early marker of inflammation and of oxidative DNA damage. Future studies should resolve the applicability of this type of assay.

7. ROLE OF ROS AND OXIDATIVE DNA DAMAGE IN CARCINOGENESIS Although the scope of this review is limited, the presented data already show that a large portion of the experimental evidence accumulated in the last several years points to ROS as active players in the carcinogenic process, by participating in initiation, promotion and progression. 7.1. ROLE OF ROS IN CELL TRANSFORMATION

Active oxygen species have been shown to be mutagenic in bacterial as well as mammalian cells (Weitzman and Stossel, 1981, 1982, 1984; Barak et al., 1983; Fulton et al., 1984; Hsie et al., 1986) and to induce a variety of other types of genetic damage, such as chromosomal aberrations and sister chromatid exchanges (Emerit et al., 1982; Weitberg et al., 1983; Phillips et al., 1984). They also include DNA strand breaks and oxidative modification of DNA bases (Weitberg et al., 1983; Zimmerman and Cerutti, 1984; Frenkel and Chrzan, 1987b; Jackson et al., 1989), which may lead to the observed sister chromatid exchanges. That ROS may be responsible for cell transformation was shown by Borek and Troll (1983), who found that CaH/10T 1/2 cell transformation by 7-radiation could be prevented by SOD. ROS generated by TPA-activated human PMNs caused malignant

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transformation of C3H/10T 1/2 cells (Weitzman et al., 1985, 1988), whereas ROS produced by hypoxanthine/xanthine oxidase caused malignant transformation of MRC5 human lung fibroblasts (Weitberg and Corvese, 1990a,b). Both transformed cell lines grew in soft agar and induced malignant tumors in nude mice. Malignant transformants of MRC5 cells showed a preponderance of a t16:18 (p13.3,q21) translocation with 13 out of 22 abnormal karyotypes showing this translocation (Weitberg and Corvese, 1990a). Hence, it is possible that this particular chromosomal abnormality represents a common marker for ROS-mediated malignant transformation. 7.1.1. Oxidative DNA Damage and Cell Transformation Recently, we analyzed MRC5 cells, which were exposed to ROS generated by human PMNs (1 hr, 3 ×/week), for the presence of oxidized DNA bases. There were increases in HMdU and 8-OHdG already within one week of exposure, which reached 10-fold and 60-fold enhancement, respectively, in the samples exposed for 5 weeks (unpublished data). Those cells were malignantly transformed within 4-5 weeks of exposure, which is at the same time as that very significant increase occurred in the levels of oxidized bases in the DNA. This increase is much higher than we found in SENCAR mouse skin treated with TPA (Wei and Frenkel, 1991a). It is possible that because MRC5 cells are human lung cells they are much more sensitive to oxidative damage than mouse epidermal cells, since lung is a target tissue for pulmonary cancer. Conversely, it is possible that the lung is a target tissue for pulmonary cancer because lung cells are very sensitive to oxidative DNA damage. 7.2. POSSIBLEPARTICIPATIONOF ABNORMALPHAGOCYTESIN CARCINOGENESIS The exposure of human lung cells to TPA-activated PMNs may be one of the models of chronic inflammation, which is known for its contribution to many types of cancer and other diseases. Another model may be based on the formation of multinucleated giant cells from normal human blood-derived monocytes that were exposed to TPA (Hassan et al., 1989). These phagocytic cells had to be kept in culture for 3 weeks prior to TPA exposure. TPA concentrations of 1 × l 0 "9 to 8 × l0 s induced cell fusion with a 30-80% fusion rate, which was still enhanced by pretreatment with a cytokine 7-interferon. This multinucleation process was presumably mediated by protein kinase C, since its inhibitor also suppressed formation of macrophage-derived giant cells. Those giant cells were also formed from the peripheral blood monocytes of patients with breast carcinoma with a higher fusion rate than those isolated from normal controls (A1-Sumidaie, 1986). Giant cells also can be formed from human PMNs. Those cells are characterized by increased myeloperoxidase activity and are found in patients with acute or chronic myeloid leukemias (Patterson et al., 1982; d'Onofrio and Mango, 1984). More recently, they were also identified in patients with acquired deficiency syndrome (AIDS) (d'Onofrio et al., 1987). Those giant cells exhibited morphological abnormalities, were abnormally matured and had irregular nuclei and a cytoplasm filled with peroxidase-containing granules. The prognoses of the AIDS patients seemed to be related to MPO levels, with higher MPO being present in patients with more advanced disease. At this point it is not clear whether abnormalities in PMNs play a role in development of the Kaposi's sarcoma that frequently occurs in AIDS patients. 7.3. ROS, OXIDIZED DNA BASESAND ONCOGENEEXPRESSION Cancer development is dependent on growth factors, genes that by being amplified or mutated escape the normal restraining controls. For example, a number of genes have been found to be expressed very rapidly after TPA treatment. These include 'immediate early' or competence genes (e.g. c-los and c-myc), which normally play a role in cell growth and differentiation (Rahmsdorf and Herrlich, 1990; Sheng and Greenberg, 1990). Expression of these normal genes is rapid and transitory and does not initially require protein synthesis. The Fos protein (a product of the c-fos gene) was found to be associated with the c-jun proto-oncogene product and together they form the transcription factor AP-1 (the activating protein) (Lamph et al., 1988; Rahmsdorf and Herrlich, 1990; Sheng and Greenberg, 1990; Gutman and Wasylyk, 1991). This heterodimer is held together by the 'leucine zipper', binds with high affinity and specificity to the consensus sequence

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-TGACTCA- and stimulates transcription of genes located nearby. This consensus sequence was first identified as a TPA-responsive element. The over expression of c-fos and c-myc in TPA-treated N M R I mouse skin was assumed to be related to hyperplasia and inflammation (Rose-John et al., 1988). However, a more direct relationship between induction of these two proto-oncogenes and TPA-mediated ROS production has been shown in cultured JB6 mouse epidermal cells (Crawford et al., 1988). Treatment of those cells by either the xanthine/xanthine oxidase system (a generator of .O2 and H202) or by TPA-induced c-fos strongly in one cell line and weakly in another, with a transient maximal induction being reached in 1 hr that diminished by 2 hr. However, c-fos mRNA increased again between 4 and 8 hr later. This temporal change is very similar to that of the TPA-mediated in vivo formation of oxidized thymidines in SENCAR mouse epidermal DNA found in our studies (Wei and Frenkel, 1991a). H M d U and dTG were maximally formed 8 hr after TPA but they also appeared as a smaller peak within 1-2 hr. Although these findings appear to be only correlative in nature, future research should show whether an over expression of these genes is dependent on oxidative DNA damage. Oxidative DNA damage includes strand breaks produced either directly or due to repair processes (Boorstein et al., 1987a,b; Teebor et al., 1988). These breaks can facilitate chromosomal translocations, which are important in carcinogenic processes and are related to the over expression of some oncogenes by juxtaposition of transcription promoters adjacent to the oncogene (Brown et al., 1986; Rabbitts, 1987; Holladay et al., 1991). The presence of oxidized bases can lead to mutations, particularly since HMdU and 8-OHdG have been shown to be mutagenic (Kuchino et al., 1987; Shirnam6 Mor6 et al., 1987; Wood et al., 1990; Shibutani et al., 1991). Some mutations and/or chromosomal translocations might also induce gene amplification, particularly mutations that lead to DNA breaks (Alitalo, 1987; Rahmsdorf and Herrlich, 1990). It is possible that oxidative DNA damage also plays a role in carcinogenesis because of the finding that deletion of an AT-rich segment of 67 base pairs (located down stream from the termination codon) results in c-fos induction of cellular transformation (Verma, 1987). Since the thymine moiety in DNA was found to be the most susceptible to the damaging effects of ionizing radiation (Cadet and T6oule, 1978; Scholes, 1983; Teebor et al., 1988), ROS generated by PMNs (which we showed to cause similar oxidative DNA base modification to that caused by v-radiation (Frenkel et al., 1986a)) or produced intracellularly are likely to damage that AT-rich segment of c-fos. Extensive formation of oxidized bases in that segment is likely to lead to their enzymatic removal (Boorstein et al., 1987a,b; Teebor et al., 1988), which may result in extensive breaks in the sugar phosphate backbone and, consequently, in the truncated c-fos with the acquired transforming activity. Another possible mechanism could be due to the binding of AP-1 to the TPA-responsive element of the collagenase gene, a process that causes stimulation of its transcription (Rahmsdorf and Herrlich, 1990). Collagenase proenzyme was shown to be activated by hypochlorous acid, a product of MPO-mediated oxidation of C1- ions by H202, where both MPO and H202 are released by TPA-stimulated PMNs (Weiss et al., 1985). Collagenase is needed for increased vascular permeability, which would lead to increased phagocytic infiltration and is thought to facilitate metastases of tumor cells (Fantone and Ward, 1982; Heppner et al., 1989). MPO is also present in monocytes, which gradually lose this enzyme during maturation into macrophages (Babior, 1984, 1987; Warren et al., 1987). Curiously, c-fos mRNA was shown to increase in monocytes during the differentiation process and was constitutively turned on in mature macrophages (Verma, 1987). The activity of tumor-associated macrophages (presumably ROS formation) was shown to increase migration of tumor cells and their metastases (Heppner et al., 1989). In contrast to c-fos, terminal differentiation switches off c-myc (Dmitrovsky et al., 1986; Rabbitts, 1987). Usually, c-myc expression is fast and transient. However, since high levels of c-myc correlate with the degree of progression, malignancy and metastasis, its gene must be aberrantly switched on (Shuin et al., 1986; Rabbitts, 1987; Cosma et al., 1989; Garte et al., 1989, 1990) to prevent terminal differentiation of cells. One of the possible times this could occur is during chronic inflammation. ROS generated under those conditions may give impetus to a chromosomal translocation of c-myc into the vicinity of actively-transcribed genes. This could result in constitutive expression of c-myc. That this may be the case is shown by an increasingly higher presence of the Myc protein in tumors. The oxidizing milieu in tumors may be provided by ROS

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generated by tumor-associated macrophages (Chong et al., 1989) (known to congregate in tumors in large amounts (up to 30% of tumor weight) (Loveless and Heppner, 1983; Heppner et al., 1989)), in addition to tumor cells themselves, which recently were shown to produce H202 in vitro (Frenkel and Gleichauf, 1991; Szatrowski and Nathan, 1991). It would be interesting to find whether the ROS-induced predominant chromosomal translocation found by Weitberg and Corvese (1990a) in human MRC5 lung cells leads to the over expression of c-myc. Another oncogene that may be related to ROS-mediated carcinogenic processes is c-abl. Weitzman et al. (1989) found polymorphism in the cytosine methylation pattern of c-abl in response to exposure of C3H / 10T cells to TPAactivated human PMNs. This polymorphism may be important because methylation of the cytosine moiety in the newly synthesized daughter DNA strand was shown to be involved in gene expression. This short overview, of the processes that link oxidant formation with induction of c-fos and c-mye and changes in the methylation pattern of c-abl, strongly points to ROS and oxidative DNA damage as potentially being responsible for the aberrant expression of these proto-oncogenes. Moreover, it shows why ROS may be involved in the early stages of tumor promotion (i.e. genetic, convertogenic step), as well as in the later stages of progression and metastasis.

8. CONCLUSIONS Although there have been many studies showing a correlation among the various ROS-producing processes and different stages of cancer development, the molecular mechanism(s) of those connections are not as yet elucidated. The most important among those correlations appear to be the following: (1) Complete carcinogens induce ROS formation and oxidative DNA damage in vitro and in vivo. (2) Antioxidants suppress cancer development, often without affecting carcinogenDNA base adduct levels. (3) Anti-tumor promoters suppress hyperplasia, PMN infiltration, H202 production and oxidation of DNA bases. (4) All of these parameters are significantly lower in the mouse strain that is resistant to TPA-mediated tumor promotion than in the sensitive strain. (5) H202 formation and oxidation of DNA bases in vitro and in vivo reflect the in vivo potencies of tumor promoting agents. (6) Tumor promoters decrease levels of antioxidant defenses. (7) Acatalatic mice develop duodenal cancer in response to treatment with H202, whereas cancer rate decreases with increasing catalase levels (Ito et al., 1984). (8) ROS derived from stimulated PMNs or enzymatically produced induce malignant cell transformation that results in cancer development in nude mice (Weitzman and Gordon, 1990). (9) Enzymatically-derived ROS or those generated by tumor promoter-activated PMNs activate proto-oncogenes involved in growth and differentiation. (10) The collagenase gene can be activated by hypochlorite produced by H202 and MPO derived from stimulated PMNs, which can lead to vascular permeability, phagocyte infiltration and tumor cell metastasis. Based on the experimental data presented above, it appears that initially tumor promoters (as exemplified by TPA) induce oxidative activation of the target cells. In response to TPA, those cells generate '02 and H202, which cause some oxidative DNA damage that is subject to repair. At the same time, chemotactic and clastogenic factors that are dependent on .O~- are also formed, since their production is SOD inhibitable. Chemotactic factors, which include the cytokines interleukins 1 and 8, mediate rapid infiltration of PMNs, a process that is inhibitable to various degrees by anti-tumor promoters. These PMNs stimulated by the residual TPA undergo oxidative burst and within 1-2 min .O~- and H202 are produced. Binding of TPA to its receptor, protein kinase C, causes translocation of protein kinase C into the membrane and its activation, which is followed in quick succession by a chain of reactions (Morel et al., 1991). These include transformation of a dormant NADPH oxidase into the active enzyme, release of arachidonic acid and its extensive oxidative metabolism to prostaglandins and leukotrienes. The signal transduction initiated by TPA is currently the subject of an intense research effort, but its scope is beyond this review. The leukotriene LTB4, produced by PMNs in response to TPA, is a potent chemotactic factor, which mediates further infiltration of PMNs into the dermis. ROS generated during the oxidative burst of PMNs induce damage to the membranes of target cells, causing lipid peroxidation, protein fragmentation and oxidative DNA damage, all of which are inhibitable by antioxidants and

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anti-tumor promoters. Oxidative modification of thymine residues in cellular DNA declines 1-2 hr after TPA treatment. However, it is further enhanced by ROS generated by the newly infiltrated PMNs and reaches maximum for 8-OHG in 6 hr and for HMU and TG in 8 hr after the initial application of TPA. The levels of all of these oxidized bases in DNA decline thereafter, but are still above the control values 24 hr after TPA treatment. When the second dose of TPA is applied, it stimulates the PMNs that in the meantime have accumulated within the dermis. Much more ROS are generated and the chemotactic factors are released. This leads to an increased migration of H202 into the target cells, potentiation of oxidative DNA damage, as well as further infiltration of PMNs followed by macrophages. Repeated applications of TPA over prolonged periods of time are likely to result in accumulation of damage in DNA, leading to chromosomal instability. Since TPA is known for its mitogenic properties, some of the unrepaired damage can cause mutations and transformation. That this may be the case is shown by the ability of TPA-activated PMNs or .O~- and the H202-producing xanthine/xanthine oxidase system to malignantly transform cells. We have found that these transformed cells contain highly elevated levels of the oxidized bases, HMU and 8-OHG, in their DNA. Treatment of epidermal cells with TPA or "02 and H202 also causes rapid induction of the competence genes that are necessary for cell growth and differentiation. The induction of those genes seems to temporally coincide with the enhanced TPA-mediated formation of oxidized DNA bases. Cumulatively then, TPA-induced oxidative activation of cells leads to ROS production and DNA damage, as well as expression of the mRNA of genes that are needed for enhanced cell growth. Moreover, this type of treatment causes an inflammatory response and infiltration of phagocytic cells, whose activation potentiates generation of ROS and genetic damage. Overall, prolonged oxidative stress also can cause systemic oxidative damage. This is shown by the presence of HMU and other oxidized bases in the DNA of human white blood cells and by the presence of anti-HMU antibodies in the sera of patients with chronic inflammatory conditions. The tumor-promoting effects of complete carcinogens have not as yet been delineated. However, it is clear that, at least initially, carcinogenic PAHs do not stimulate PMNs, as measured by H202 production. Nevertheless, it cannot be ruled out that the PAH metabolite(s) might activate PMNs and it may even be likely because DMBA and B(a)P cause inflammation in the mouse skin. PAHs are metabolized by target cells to various products, some of which bind to DNA and form adducts. Other metabolites (i.e. quinones) can be produced if the PAHs have an appropriately low potential to form carbocations and both B(a)P and DMBA do. Those quinones can undergo redox cycling, a process that generates substantial amounts of ROS, which might enhance peroxidatic formation of initiating agents (i.e. diol epoxides) that are considered to be ultimate carcinogenic metabolites. Those ROS could also induce formation of chemotactic factors by epidermal cells, which would cause infiltration of phagocytes. That this reasoning may be correct was shown by a persistent PMN presence over a period of approximately 3 weeks after the tenth application of DMBA. At the same time, HMdU levels were still increasing for at least 48 hr in the DNA of epidermal cells isolated from the DMBA treated mice, while in the DNA from TPA-treated mice, HMdU reached its maximum within 24 hr. It appears then that the continued DMBA metabolism delivers products that can still increase HMdU formation, presumably due to the continued production of ROS by the DMBA metaboliteactivated phagocytic cells. Future work should lead to elucidation of the roles of ROS and oxidative DNA damage in carcinogenesis. Especially important will be experiments that will allow determination of whether anticarcinogenic agents inhibit PAH-induced formation of oxidized bases in the DNA of target cells as they inhibit PAH-mediated carcinogenesis. If they do, then a more common and frequent use of anticarcinogenic agents, many of which occur in a variety of foods, may lead to the prevention of at least some types of cancer.

Acknowledgements--I would like to thank past and present members of my laboratory for their superb work and dedication, especially K. Chrzan, Z. Zhong and J. Karkoszka and Drs Wei and Bhimani. I appreciate the willingness of the many scientific colleagues to share with me the results of their as yet unpublished work. They include Drs Cavalieri, Conney, Demple, Fiala, Fisher, Kasprzak, Kensler, Loeb, Malejka-Giganti,

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Robertson and Wetterhahn. I am also greatly indebted to Drs C. Klein and A. Stern for their critical reading of the manuscript and to Dr W. Troll for his encouragement in all of my endeavors. This work was supported in part by grants number CA 37858 and CA 49798 from the National Cancer Institute and by grants number 1 P42 ES 04895 and ES 00260 from the National Institute of Environmental Health Sciences.

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