7,12-dimethylbenz[a]anthracene induces oxidative DNA modification in vivo

Free Radical Biology & Medicine, Vol. 19, No. 3, pp. 373-380, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

Pergamon

0891-5849(95)00046-1

Brief Communication 7,12-DIMETHYLBENZ[A]ANTHRACENE INDUCES OXIDATIVE DNA M O D I F I C A T I O N IN V I V O

KRYSTYNA FRENKEL, *t$ LIHONG WEI,* and HUACHEN WEI *§ Departments of *Environmental Medicine and tPathology, and ~Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, NY, USA (Received 3 November 1994; Revised 8 March 1995; Accepted l0 March 1995)

Abstract Initiation and promotion are major stages in the multistage carcinogenesis process. Formation of initiating carcinogenDNA base adducts leads to heritable genetic changes, but the tumor-promoting events induced by complete carcinogens have not, as yet, been elucidated. Oxidant production and oxidative DNA damage induced by phorbol esters (i.e., 12-O-tetradecanoylphorbol-13-acetate) are associated with tumor promotion, while antioxidants and inhibitors of oxidative DNA damage suppress promotion and carcinogenesis. Our goal was to establish whether a carcinogen that requires oxidative metabolism for its activity can also induce oxidant production and DNA base oxidation. We found that topical treatment of SENCAR mice with 7,12dimethylbenz[a]anthracene, which induces tumors in 40-50% of the mice, also causes hydrogen peroxide production and formation of oxidized bases (i.e., 8-hydroxyl-2'-deoxyguanosine and 5-hydroxymethyl-2'-deoxyuridine) in epidermal DNA. The levels of oxidized bases were of comparable magnitude to those mediated by the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The oxidized bases persisted over several weeks in epidermal DNA. These oxidative events appear to be temporally associated with inflammatory responses that include edema and polymorphonuclear leukocyte infiltration, which remained elevated over longer periods of time and at higher levels than those induced by phorboi ester. Because these processes are usually associated with tumor promotion, our results support the conjecture that oxidative events may be involved in what is operationally referred to as the tumor promotion process by 7,12-dimethylbenz[a]anthracene. Keywords---Free radicals, Carcinogenesis, Tumor promotion, Oxidative stress, Inflammation, Polycyclic aromatic hydrocarbons, Oxidized DNA bases

INTRODUCTION

it is formation of hydrogen peroxide (H202, which is itself a tumor promoter) that best correlates with tumor promotional processes in general and oxidation of DNA bases in particular. 5-7 Although there are extensive antioxidant defenses, as well as enzymatic removal and repair of oxidative DNA damage in mammalian cells,s-~° some of those oxidized DNA base derivatives were shown to be mutagenic. IH3 They include 5-hydroxymethyl-2'-deoxyuridine (HMdUrd) and 8-hydroxyl-2'-deoxyguanosine (8-OHdGuo), an oxidized dThd and dGuo, respectively. It seemed important to establish the mechanism of action of the polycyclic aromatic hydrocarbon (PAH) by finding whether complete carcinogens (such as 7,12-dimethylbenz[a]anthracene (DMBA)), which by definition initiate events leading to cancer and induce progression of benign to malignant tumors, ~4-t6 also cause occurrence of oxidative processes, which are considered hallmarks of tumor promotion,z~7-~9This knowledge could allow protective measures to be designed that might possibly be incorporated into the human diet, because tumor

The carcinogenic process has been shown to involve chronic inflammation,L2which is mediated by stimulation of infiltrating phagocytic cells (i.e., polymorphonuclear leukocytes (PMNs) and macrophages). That stimulation causes an oxidative burst and generation of reactive oxygen species (ROS). 2-4 Of those ROS, Address correspondence to: Krystyna Frenkel, Department of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016-6451, USA. This work was supported by Grant CA 37858 from the National Cancer Institute (its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute), and by Grant ES 00260 from the National Institute of Environmental Health Sciences. Part of this work was presented at the Annual Meeting of the American Association for Cancer Research (Wei, H.; Frenkel, K. 7,12-Dimethylbenz[a]anthracene (DMBA)-mediated in vivo induction of oxidative events and oxidative DNA damage in SENCAR mice. Proc. Am. Assoc. Cancer Res. 33:179; 1992). Current address: Department of Environmental Health Sciences, University of Alabama at Birmingham, School of Public Health, Birmingham, AL 35294-0008, USA. 373

K. FRENKELet al.

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promotion can be interfered with much more readily than the initiation of carcinogenesis.2"7,17-2° DMBA, a representative PAH, was chosen for this study because previously we showed that in vitro exposure of rat liver microsomes to any of several carcinogenic PAHs (i.e., DMBA, benzo[a]pyrene (B[a]P), or 3-methylcholanthrene) causes dose- and time-dependent production of 1-1202, an oxidant most closely associated with tumor promotion, while the noncarcinogenic pyrene was totally inactive. TM Moreover, oxidized bases were present in DNA exposed to B[a]P-treated hepatic microsomes at levels 4 - 6 times higher than in controls, and their formation was suppressed by catalase, thus pointing to the involvement of H202.2~Because metabolism of PAHs by liver microsomes was shown to differ from that carried out by intact cells, 22"23we performed a number of experiments using a whole animal system. SENCAR mice were used in these experiments because this mouse strain is sensitive to tumor promotion by 12O-tetradecanoylphorbol-13-acetate (TPA) as well as to complete carcinogenesis by DMBA. 24 Moreover, we previously studied effects of TPA and other phorbol ester-type promoters on oxidative stress responses of this m o u s e s t r a i n . 6'25-27 In experiments described in this report, we compared responses of SENCAR mice treated either with DMBA or TPA by measuring inflammation (edema, hyperplasia, and PMN infiltration) and H202 production in the skin, and formation of oxidized nucleosides in the epidermal DNA.

MATERIALS AND METHODS

Materials SENCAR mice were purchased from Biological Testing, NCI (Frederick, MD), DMBA from Aldrich Chemical Co., Inc. (Milwaukee, WI), while TPA, 2'deoxyribonucleosides (dGuo, dAdo, dCyd, dThd, and HMdUrd), catalase, myeloperoxidase (MPO), horseradish peroxidase, and various other reagents were from Sigma Chemical Co. (St. Louis, MO). ASAP genomic isolation columns, and enzymes (RNase A, proteinase K, DNase I, nuclease P1 and alkaline phosphatase) needed for DNA purification and hydrolysis were obtained from Boehringer-Mannheim Biochemical (Indianapolis, IN). HPLC-grade acetonitrile, acetone, and reagents were purchased from Fisher Scientific (Springfield, NJ). [3H]Acetic anhydride (sp. act. 50 mCi/mmol) was purchased from DuPont/New England Nuclear (Wilmington, DE). The following marker compounds were synthesized and purified in this laboratory, as previously described28: 8-OHdGuo, its acetates, and HMdUrd triacetate.

Treatment of animals SENCAR mice (6-7-week old females) were divided into three groups of 40 each, acclimated for 1 week, 29 and shaved 48 h before topical treatment (Tx) with DMBA (100 nmol/Tx) or TPA (6.5 nmol/Tx) dissolved in acetone (200 pl/Tx), or only with acetone (2 times/week for 5 weeks). After Tx termination, the mice in each group were divided into two subgroups, one for observation of tumor development and the other sacrificed 1 h (Point "0"), or l, 2, 7, 14, 21, or 35 days after the last (tenth) Tx, and used for determination of various endpoints. After the last Tx, tumors were counted once/week for 10 weeks (15-week total experimental period), and only those > 1 mm2 in diameter were considered. The first papillomas appeared in the skin during Week 5 of the DMBA treatment and were counted within 1 week from Tx termination. Skin punches (1.227 cmE/punch made with a cork borer), which included epidermal and dermal cells and connective tissue, were used for determination of edema (increases in weights of experimental as compared to control skin punches are expressed in mg/cm2), their MPO content and H202 accumulation.26'29 The remaining skin (two mice/point) was used for quantitation of oxidized DNA bases.

PMN infiltration: quantitation of myeloperoxidase Although MPO is present in PMNs and monocytes, the latter lose MPO as they mature into tissue macrophages, 3° and therefore MPO is used as a measure of PMN infiltration into mouse skin. MPO was determined in whole skin punches as previously described. 29 Briefly, skin punches were placed in 50 mM potassium phosphate (pH 7.0) buffer containing hexadecyltrimethyl ammonium bromide, minced, homogenized while being kept ice-cold, and centrifuged. MPO was determined in supernatants, using a 4-aminoantipyrine/ phenol solution as the substrate for MPO-mediated oxidation by H202, and changes in absorbance at 510 nm (A5~0) were recorded. One unit of MPO activity is defined as that which degrades 1 #mol H202/min at 25°C. Data are presented as mean values (in units/cm2) ± SE from two independent experiments with a total of 6 - 8 determinations per point.

Determination of H202 The method used is based on horseradish peroxidase-mediated oxidation of phenol red5'31 by the H202 present in mouse skin. H202 was measured in skin punches, consisting of epidermal and dermal cells, with the connective tissue removed by scraping. 26 Briefly,

DMBA-induced oxidative DNA damage skin punches were immediately placed in ice-cold 50 mM phosphate buffer, pH 7.0, containing 10 mM sodium azide as the catalase inhibitor, minced, homogenized, and centrifuged at 4°C. Supernatants were promptly stored at - 8 0 ° C for no longer than 7 days before H202 determination. These storage conditions had virtually no effect on H202 quantitation. H202 was measured in 0.5 ml of the supernatant, which had been mixed with 0.5 ml of the same buffer containing phenol red (100 #g) and horseradish peroxidase (50 #g) and incubated for 10 min at room temperature. Absorbance of solutions alkalinized with NaOH was determined at 610 nm. Although other oxidants present in the supernatants could also contribute to the increase in A61o, we previously determined that 6 0 - 8 5 % of oxidants present in the mouse skin-derived preparations is catalase inhibitable. We also found that expressing results as nmol/cm 2 is more appropriate as a measure of treatment-induced changes than nmol/mg protein, because both H202 and protein levels are changed in carcinogen-treated skin, whereas the surface area remains about the same. 26 For these reasons, data are presented as mean values in nmol H202/cm 2 _ SE obtained in two independent experiments and 6 - 8 determinations.

Quantitation of oxidized bases Immediately after sacrificing the treated mice and removing skin punches for determination of edema, MPO, and H202, the remaining dorsal skin (excluding tumors, two mice/point) was immersed in hot water (60°C) for 30 s and immediately placed in ice-cold water, 29 which allowed easy removal of the epidermal layer within a few minutes. Epidermal cells were suspended in phosphate buffer, homogenized, and centrifuged, and the pellet used for DNA isolation and purification on ASAP columns. 26'29Purified DNA was precipitated with isopropanol, and the precipitate was washed twice with 70% ethanol, dried under nitrogen, and stored at - 8 0 ° C until analysis. 29 DNA ( ~ 100/zg/ analysis, equivalent of ~0.35 #mol bases) was enzymatically digested to nucleosides, the enzymes were removed by acetone precipitation, and the supernatant was evaporated under reduced pressure. 28'29 The residue was dissolved in a small amount of HPLC-grade water (all solvents used in the isolation of DNA were HPLC grade), filtered through a 0.2 /zm membrane and nucleosides separated by HPLC (Beckman, Model 344) on an octadecylsilane (ODS) column (Altex, 25 × 1.0 cm i.d.; 5 /zm particle size) in the absence of any external markers. Normal nucleosides present in DNA hydrolyzates were quantitated as previously described, 28 using integrated peak areas (6300 WGS AT&T computer with ChromatoGraphics software

375

from Beckman) and standard calibration with marker nucleosides. Fractions having retention times known to elute 8-OHdGuo and HMdUrd were combined, evaporated to dryness, dissolved in acetoniWile, and acetylated with [3H]acet;.c anhydride (10/zmol; with a 1:10 ratio of tritiated to nonradioactive acetic anhydride, sp. act. 9900 cprn/nmol triacetate) in the presence of the catalysts 4-dimethylaminopyridine and triethylamine. 28 Nonradioactive marker triacetates of HMdUrd and 8-OHdGuo were added to DNA-derived tritiated acetates and, after concentration and filtration through 0.2 /~m membranes, analyzed by HPLC. 28"29 Results are presented as mean values of a number of oxidized bases per 104 normal nucleosides _ SE obtained in two independent experiments. This assay can reliably measure 5 - 1 0 pmol oxidized nucleosides, 28 and therefore 100/zg DNA used for each point provides sufficient material for the analysis.

Statistical analysis of data Results are presented as mean values _+ SE of two independent long-term experiments, unless only one is depicted (as in Fig. 2B). MPO and H202 values are means of 6 - 8 determinations. Statistical significance was determined according to the Student's t-test with * being p < .05, ** p < .005, and *** p < .001 in DMBA vs. TPA; +++ being p < .001 in DMBA vs. acetone; while ++ represents p < .01 between the H202 formation 14 and 21 days after the last (tenth) Tx. RESULTS The dorsal skin of both DMBA- and TPA-treated mice showed marked inflammatory responses, including edema, phagocytic infiltration, and hyperplasia. Although at the end of treatment and for the following 3-week period edema was comparable, it was significantly higher in DMBA- than in TPAtreated mice 35 days after the last Tx (Fig. 1A), at which time the latter returned to the value of acetone controls. Similarly, MPO leveis were comparable for 2 days after the last Tx, but at 7, 21, and 35 days after Tx 10, MPO levels also were significantly more elevated in the skin of DMBA- than of TPA-treated mice (Fig. 1B). MPO levels in acetone-treated mouse skin were negligible (not shown). DMBA-induced H202 accumulation (Fig. 2A) was initially (1 h after Tx 10, Time " 0 " ) comparable to that induced by TPA. After 24 h it was significantly higher than in the acetone-treated controls, but was much lower than that mediated by TPA at 1 to 14 days after Tx, at which time ( 2 - 1 4 days) it was at the level of acetone controls (not shown). Lower H202 levels in

376

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TIME AFTER LAST T×(D) Fig. 1. DMBA- and TPA-mediated inflammatory responses of SENCAR mice 5 weeks after beginning treatments (two Tx/week). (A): Edema. There were virtually no variations in punch weights among acetone-treated control mice sacrificed at six different time points after Tx 10, with a mean weight of 61.0 + 3.2 mg/cm2, shown as a straight line on the graph. Results are presented as mean values (mg/cm2) + SE of two determinations. (B): MPO as a measure of PMN infiltrationinto mouse skin. Data are presented as mean values (MPO units/cm2) _+ SE from two independent experiments with a total of 6-8 determinations per point. Statistical significance of effects of DMBA vs. TPA was determined by Student's t-test, and is depicted as *p < .05, **p < .005, and ***p < .001. DMBA Tx (O), TPA Tx (&), Acetone Tx (...); with Tx representing treatment.

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i4 D M B A - as o p p o s e d to T P A - t r e a t e d s a m p l e s c o u l d be p a r t i a l l y a t t r i b u t e d to c o n s u m p t i o n o f HzO2 d u r i n g o x i d a t i v e m e t a b o l i s m o f D M B A (such as peroxidatic o x i d a t i o n o f diols to diol epoxides). 32-34 It c o u l d also be due to a s u b s t a n t i a l release o f M P O b y s t i m u l a t e d P M N s a n d HzO2 u t i l i z a t i o n b y M P O - m e d i a t e d oxidative processes. 2-4"32 In contrast, TPA is not metabolized, the H202 generated is not utilized for the metabolic processes, and M P O release is low.E-4Curiously, after returning to control levels, DMBA-induced H202 production was significantly elevated again within 21 in comparison to 14 days past the last Tx (Fig. 2A). This enhancement in H202 production coincided with the period of the vigorous linear development and growth of

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TIME AFTER LAST TX(D) Fig. 2 H202 formation in mouse skin (A) and tumor development (B) in SENCAR mice 5 weeks after beginning treatments (two Tx/ wk). (A): H2Oz was measured in skin punches with connective tissues removed by scraping.29 Data are presented as mean values (nmol H202/cm2) -+ SE obtained in two independent experiments and 6-8 determinations. Statistical significance was determined by Student's t-test, with *** being p < .001 in DMBA- vs. TPA-, and +++ being p < .001 in DMBA- vs. acetone-treatedmouse skin, while ++ represents p < .01 of 21 vs. 14 days in DMBA4reated mouse skin. (B): Representativeexperiment (20 mice,rEx) in which 50% of the DMBA-treated mice developed tumors by the end of the experiment with 1.9 tumors/mouse. In another experiment, 40% of the mice developed tumors. At the same time, TPA- or acetone-treated mice (20 mice/group) did not develop any tumors, although some TPA-treated mice showed locus proliferation. DMBA Tx (O), TPA Tx (A), Acetone Tx ([]); Tx represents treatment.

DMBA-induced oxidativeDNA damage DMBA-induced minors exhibited spreading, bleeding, and necrosis (not shown). Both DMBA and TPA induced enhanced formation of the oxidized bases 8-OHdGuo and HMdUrd, which persisted in the epidermal DNA for up to 35 days after the last Tx, as shown in Figure 3. Levels of 8-OHdGuo in DNA of TPA-treated mice (Fig. 3A) gradually declined and within 14 days past Tx 10 nearly reached control levels. The 8-OHdGuo in DNA of DMBAtreated mice reached its lowest level within 7 days, at which time it was equal to that mediated by TPA but, in contrast to TPA, it remained significantly elevated until the end of the observation period (Fig. 3A). Also, HMdUrd (Fig. 3B) levels were higher in DMBA- than in TPA-treated samples, and then gradually declined but were still 2-fold higher at the end of the experiments than those of acetone controls. Although 8-OHd-

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377

Guo and HMdUrd were present in higher amounts in the epidermal DNA of DMBA- than in TPA-treated mice, those differences were not statistically significant. Both 8-OHdGuo and HMdUrd levels in epidermal DNA of DMBA- and TPA-treated mice were significantly elevated in comparison to acetone controls. Interestingly, there were two peaks of enhanced oxidized DNA base formation; one in 2 days (early) and the second 21 days (late) after the last Tx with DMBA or TPA, with those in DMBA-treated samples being more pronounced. The late peaks of 8-OHdGuo and HMdUrd formation coincided with elevated production of H2Oz by DMBA-treated mice (Fig. 2A) and with vigorous tumor growth (Fig. 2B).

DISCUSSION Previous studies have shown that TPA and some other phorbol ester-type mouse skin tumor promoters produce ROS and oxidative modification of DNA bases, which are strongly associated with promotion.2,6,17-19.34 Antitumor promoting agents such as S arcophytol A (from a marine coral), CAPE (caffeic acid phenethyl ester, from the propolis of honeybee hives), and EGCG ((-). epigallocatechin gallate, polyphenol from green tea) prevent TPA-induced ROS production as well as oxidative DNA damage. 2'6'7'2627'37-4° The present study demonstrates that DMBA, a complete skin carcinogen, induces substantial oxidative effects in vivo that are similar to those of TPA. A prominent effect is oxidative modification of DNA bases, which occurs during the same time period that baseDMBA adducts are formed. Major events associated with tumor promotion are of longer duration after multiple DMBA treatments than those induced by TPA. These include enhancement of inflammatory responses (as measured by edema), neutrophil infiltration (quantitated by MPO), and oxidation of bases in the epidermal DNA (determined by HMdUrd and 8-OHdGuo) obtained from treated mouse skin. Many other types of oxidative DNA base modification in addition to 8-OHdGuo and HMdUrd may be induced by oxidative stress, 2'37 but these have not been examined. That ROS and oxidative DNA base formation may be a more general tumor-promoting effect of complete carcinogens is indicated by formation of these and/or other oxidized bases in DNA of cells exposed to various carcinogens, which include 4-nitroquinoline N-oxide, B[a]P, ionizing and UV radiations, 2-nitropropane, KBrO3, diethylstilbestrol, diethylnitrosamine, and 2acetylaminofluorene.Z41-44 The actual source of the oxidants causing oxidative DNA base modification is not as yet known. However,

378

K. FRENKELet aL

stimulated PMNs can produce substantial amounts of various ROS, with H202 being most important because it is the only ROS that can easily reach nuclear DNA because of its facile crossing of plasma and nuclear membranes. Moreover, H202 is a precursor of both hydroxyl radical-like species as well as a singlet oxygen, which are known to cause oxidative modification of DNA bases. 2'3"41 Hydroxyl radicals mediate formation of both HMdUrd and 8-OHdGuo. 2 In contrast, only 8-OHdGuo can be produced by singlet oxygen. 4~ Therefore, HMdUrd can be considered a measure of hydroxyl radical-induced oxidation, while 8-OHdGuo can be considered a measure of both hydroxyl radicals and singlet oxygen. It is known that depending on the activating stimulus, the amount of MPO released by PMNs may v a r y . 2-4 For example, TPA causes much lower MPO release than does fmet-leu-phe (fMLP). Thus, when more MPO is released, a higher proportion of H202 can be diverted to the MPO-mediated oxidative processes that consume H202. This would lead to seemingly lower measurable H202. Hence, our method does not measure H202 formation but its accumulation, which represents a net difference between its formation and utilization. Because DMBA does not activate PMNs (unpublished data), it must be its metabolite(s) that is responsible for PMN stimulation. It is therefore possible that apparent differences between TPA- and DMBA-mediated H202 accumulation over the period of time after the last treatment are at least partially due to differences in the levels of released MPO. This aspect will have to be resolved in the future. The postulated higher amount of released MPO can also participate in processes that may result in an enhanced formation of singlet oxygen. 2-4 This process occurs when MPO catalyzes H202-mediated oxidation of C1- ions to hypochlorite, a potent oxidizing agent, which can be reduced by an excess H202 back to CI- ions with a concomitant evolution of singlet oxygen, and thus can account for further consumption of the originally generated H202. It was estimated that the DNA of cells treated in culture with B[a]P contains an ~,20-fold higher level of oxidized bases than B[a]P-base adducts. 42 Our results show that in vivo Tx with a PAH mediates formation of at least 4 - 1 0 oxidized bases per 104 normal bases, while the level of adducts generally is in the order of 1 per 106--107 bases. Although there are numerous antioxidant defenses in mammalian cells, which also possess repair enzymes that recognize oxidized bases in DNA, 2'8-t° the presence of such extensive oxidative damage might impair the removal of carcinogen-DNA adducts and be responsible for their known persistence. 45'46For example, a pronounced inhibition of uracil removal by uracil DNA-glycosylase was shown in

the presence of bulky adducts. 47'4~This points to modulating effects of one type of DNA modification on the repair of another. Although mammalian cells possess glycosylases recognizing oxidized bases in DNA, these enzymes are not as efficient as uracil DNA-glycosylase. 8-~° Moreover, it has been shown that oxidized bases accumulate in DNA under inflammatory conditions even in the absence of base-carcinogen adducts, the presence of which would likely further inhibit removal of oxidized bases. Some oxidized nucleosides (i.e., HMdUrd and 8-OHdGuo) themselves can be mutagenic. 2'~t-~3 They were also shown to cause hypomethylation, 49'5° and thus, may have an effect on gene expression. 51 Therefore, those enhanced levels of oxidized bases in DNA in conjunction with DMBA-DNA base adduct formation could have a pronounced effect on the heritable processing of DNA modifications. Recently, it was shown that TPA-induced oxidative stress 34 as well as oxidants generated due to the action of MPO released by PMNs 32 cause enhanced metabolism of the 7,8-di01 of B[a]P to the initiating carcinogenic diol epoxide, and potentiates its binding to DNA of epidermal cells in vivo. It is not known as yet whether and how presence of oxidants influences P450-mediated metabolism of the parent PAH to 7,8 diol. Therefore, it is uncertain whether formation of 7,8 diol or of diol epoxide is a rate-limiting step. Nonetheless, this concomitant DNA modification (due to formation of base-adducts and oxidized bases) and the likely decrease in DNA repair capacity are probably responsible (at least in part) for the stronger carcinogenic potential of complete carcinogens 52 than when animals are treated with a subcarcinogenic dose followed by a tumor promoter in a two-stage carcinogenesis model. Our results may also explain observations made some time ago, which showed that antioxidants inhibit experimental carcinogenesis induced by PAHs, 53-56 although they do not always decrease levels of DNA base-PAH adducts. 55 In summary, data presented in this communication show that DMBA, a potent complete chemical carcinogen, causes oxidative stress and oxidative modification of DNA in vivo at least as pronounced as does TPA, a typical tumor promoter. Moreover, it causes formation of the same types of oxidized DNA bases as does ionizing radiation, 2'57 an archetypical free radical-generating complete carcinogen with a potent tumor-promoting activity. Several applications of DMBA provide an added complexity to interactions between concomitantly formed what is operationally known as "initiating" (carcinogen-DNA base adducts) and "promoting" (oxidized bases) lesions, which cannot be accounted for in the two-stage model of a subcarcinogenic dose of an initiator subsequently followed by

DMBA-induced oxidative DNA damage

multiple doses of a promoter. These concurrent interactions of "initiating" and "promoting" processes then may be the key to complete chemical carcinogenesis. Acknowledgements - - We are grateful to Drs. Belman, Snow,

and Troll for their helpful discussions and critical reading of the manuscript.

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ABBREVIATIONS

B[a]P--benzo[a]pyrene DMBA-- 7,12-dimethylbenz[a]anthracene HMdUrd--5-hydroxymethyl-2'-deoxyuridine H202--hydrogen peroxide 8-OHdGuo--8-hydroxyl-2'-deoxyguanosine MPO-- myeloperoxidase PAH--polycyclic aromatic hydrocarbon PMNs--polymorphonuclear leukocytes ROS--reactive oxygen species TPA-- 12-O-tetradecanoyl-phorbol- 13-acetate Tx--treament