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

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(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 (