Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

(1987)

%$337-346

Role of Inflammatory Cells in the Metabolic Activation of Polycyclic Aromatic Hydrocarbons in Mouse Skin THOMAS

Division

W. KENSLER, PATRICIA A. EGNER, K. GREGORY MOORE, BONITA G. TAFFE, LORRAINE E. TWERDOK. AND MICHAEL A. TRUSH of Experimental Johns Hopkins

Pathology and Toxicology, Department of Environmental School of Hygiene and Public Health, Baltimore, Maryland

Received

November

24,1986:

accepted

May

Health Sciences, 21205

4. 1987

Role of Inflammatory Cells in the Metabolic Activation of Polycyclic Aromatic Hydrocarbons Mouse Skin. KENSLER, T. W., EGNER, P. A., MOORE, K. G., TAFFE, B. G., TWERDOK, L. E., AND TRUSH, M. A. (1987). Toxicol. Appl. Pharmacol. 90, 337-346. Oxidants, such as those generated by activated polymorphonuclear leukocytes (PMNs) during inflammation, have been implicated in the metabolic activation of procarcinogens to their ultimate carcinogenic form. In this study we examined the effect of inflammation on the metabolic activation of (?)tranr-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8dihydrodiol) to a covalent binding species in mouse epidermis. Interaction of BP 7,8-dihydrodiol with 12-O-tetradecanoylphorbol- 13-acetate (TPA)-stimulated murine leukocytes resulted in the generation of both a chemiluminescent intermediate and one that covalently bound to the DNA of cocultured epidermal keratinocytes. Topical treatment of mouse skin with TPA led to an influx of PMNs into the skin beginning several hours after application. Myeloperoxidase activity, a marker for neutrophils, increased 1Sfold in the skin by 16 hr after TPA treatment. Dual applications of TPA at both 16 hr before and concurrently with administration of [‘H]BP 7,8dihydrodiol led to a 50% enhancement of the level of carcinogen that was covalently bound to epidermal DNA. However, a single application of TPA, either 16 hr before or concurrently with BP 7,8-dihydrodiol administration, had no enhancing effect, suggesting that both initial recruitment of PMNs into the skin and subsequent stimulation of oxidant production by the PMNs were required to enhance carcinogen binding. By contrast, no enhancement of benzo[a]pyrene binding was observed by TPA treatments in vivo. However, TPA-stimulated neutrophils did not activate this procarcinogen to a chemiluminescent metabolite in vitro. These results suggest that oxidants generated by metabolically stimulated PMNs can activate penultimate polycyclic aromatic hydrocarbons, such as BP 7,8dihydrodiol, to potentially genotoxic metabolites in vivo and further define a role for inflammation in carcinogenesis. 0 1987 Academic Press, Inc. in

The actions of most mutagens or carcinogens can be attributed to biotransformation from relatively inert chemicals to highly reactive metabolites capable of interacting with biomolecules (Miller, 1970; Conney, 1982). It is generally acknowledged that ultimate carcinogenic metabolites are electrophilic reactants and that the initiation of carcinogenesis is closely linked with the formation of specific carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a well-studied polycyclic aromatic hy337

drocarbon that has been particularly useful in dissecting the relationship between metabolic activation and chemical carcinogenesis. One pathway for activation of BP involves a specific sequence of reactions resulting in the generation of a bay region diol epoxide that is mutagenic, elicits the transformation of cells in culture, and is carcinogenic in several tissues in vivo, including the skin (Levin et al., 1977; Conney, 1982). This diol epoxide can be formed from the penultimate metabolite 0041-008X/87

$3.00

Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tram - 7,8 - dihydroxy - 7,8 - dihydrobenzo[a] pyrene (BP 7,8-dihydrodiol) through the catalytic actions of microsomal cytochrome P450 (Gelboin, 1980) by an oxidant generated during lipid peroxidation (Dix and Marnett, 1983) or by the peroxidase component of prostaglandin synthetase (Marnett, 198 1). Eling et al. (1986) have provided evidence that the oxidation of (+)-BP 7,8-dihydrodiol can occur through peroxyl radical- and monooxygenase-dependent pathways in mutine keratinocytes. Battista et al. (1984) have suggested that a peroxyl radical mediated the epoxidation of BP 7,8-dihydrodiol observed during lipid peroxidation, whereas a cytochrome P-450-0x0 complex was implicated in this activation by the mixed function oxidase system. Concomitant with this metabolism of BP 7,8-dihydrodiol is the formation of a 9, IO-dioxetane intermediate which decomposes with the emission of photons, as indicated by chemiluminescence (Hamman et al., 1979, 198 1). The interaction of BP 7,8dihydrodiol with singlet oxygen generated by chemical systems also results in chemiluminescence with an emission spectrum identical to that observed when it is metabolized by rat liver microsomes (Seliger et al., 1982). Other polycyclic aromatic hydrocarbons, such as chrysene and 3-methylcholanthrene, can also be metabolically activated by singlet oxygen (McCoy and Rosenkranz, 1980). Increased generation of reactive oxygen species such as the superoxide anion and hydroxyl radical is characteristic of activated inflammatory cells, including polymorphonuclear leukocytes (PMNs) (Allen, 1980; Weiss and LoBuglio, 1982). Through the catalytic actions of myeloperoxidase from PMNs the reactivity of superoxide anion and hydrogen peroxide is amplified by the generation of sinsinglet oxygen (Allen, 1980; Weiss and LoBuglio, 1982). The oxidants generated by metabolically stimulated PMNs can serve to activate BP 7,8-dihydrodiol to genotoxic and chemiluminescent metabolites through myeloperoxidase-dependent pathways. Structure-activity studies have demonstrated a

ET AL.

strong correlation between PMN-mediated activation of polycyclic aromatic hydrocarbons to chemiluminescent products and the generation of metabolites that elicit mutagenesis in Salmonella typhimurium strain TA 100 and cause sister chromatid exchanges in cocultured V-79 fibroblasts (Trush et al., 1985, 1986). PMN-mediated activation of other carcinogens, such as aflatoxin B, , N-hydroxyacetylaminofluorene, and benzidine, has also been observed in vitro (Trush et al. 1986; Tsuruta et al. 1985). Such an activation mechanism could provide a partial explanation as to how neoplasms often develop at sites of ongoing inflammation; however, all of the available evidence has come from in vitro experimentation. It is the purpose of this study to characterize a model system for examining the effects of inflammation on the activation of chemical carcinogens in vivo. To our knowledge, the results presented in this report provide the first direct evidence that an inflammatory state may enhance the metabolic activation of a chemical carcinogen in a target tissue in vivo. METHODS Chemicals. Tritiated BP (50.5 Ci/mmol) was purchased from Amersham (Arlington Hts, IL) and tritiated BP 7,8-dihydrodiol (314 Ci/mmol) from Midwest Research Institute (Kansas City, MO). Hexadecyltrimethylammonium bromide, o-dianisidine dihydrochloride, protease, paminosalicylic acid, and hydroquinidine were obtained from Sigma (St. Louis, MO) and 12-0tetradecanoylphorbol- 13-acetate (TPA) from L. C. Services (Wobum, MA). All other chemicals were of the highest grade available commercially. Animals. Female CD- 1 mice (7-9 weeks of age) were obtained from Charles River Breeding Laboratory (Wilmington, MA). Backs of mice were shaved with surgical clippers 2 days prior to experimental use and only those mice not exhibiting hair regrowth were used. All chemicals were applied topically to the shaved area in 0.2 ml of acetone. Histology. Tissues were excised and fixed in 4% paraformaldehyde buffered with 2% calcium acetate for 2 hr at room temperature (Crouch and Thaete, 1985). Samples were then washed in 0.05 M potassium phosphate, pH 7.2, for 48 hr at 4°C followed by sequential dehydration in 50,70, and 95% ethanol (all containing 2.5% glyc-

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erol) for 90 min each as described previously (Moore et al., 1986). Skin samples were then embedded in glycolmethacrylate using the JB-4 kit from Polysciences, Inc. (Warrington, PA), sectioned to 4 pm thickness, transferred to glass slides, and stained using a Giemsa solution. Stained skin sections were examined microscopically for the presence of inflammatory cellular infiltrate. Cellular infiltrate was morphologically characterized by selective granule staining of cell types and nuclear pattern. Assay ofmyeloperoxidase. Mice were killed by cervical dislocation and dorsal skins were removed. Myeloperoxidase was extracted from punches (obtained using a 22mm diameter cork borer) of dorsal skin by homogenization of minced tissue with a Polytron homogenizer in 5 ml of 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0. The specimens were freeze-thawed and sonicated three times at 4°C. Suspensions were then centrifuged at 15,OOOgfor 20 min. Myeloperoxidase was assayed by admixing 0.1 ml of the resultant supematant with 2.9 ml of 50 mM potassium phosphate, pH 6.0, containing 0.167 mg/ml of odianisidine dihydrochloride and 0.0005% hydrogen peroxide and measuring the change in absorbance at 460 nm with a Beckman DU-7 spectrophotometer (Bradley et al., 1982). One unit of myeloperoxidase activity was defined as that degrading 1 gmol of peroxide per minute at 25°C. Approximately 8.6 X IO6 murine PMNs isolated from peripheral blood (Kensler and Trush, 198 1) yielded 1 unit of activity. Isolation ofcells. Peritoneal PMNs were isolated from female CD-l mice given a 2 ml ip injection of 10% glycogen in saline 16 hr prior to termination. Cells were subsequently harvested as described by Pember and Barnes (1983) and centrifuged at 1SOg for 10 min at 4°C. Contaminating erythrocytes were lysed by adding cold 0.155 M NH,Cl/O.Ol M KHCO,/O. I mM EDTA, pH 7.4. PMNs were washed, resuspended in Hanks’ balanced salt solution (HBSS), and counted on a hemacytometer. Preparations typically contained 50-70% leukocytes; the remaining cells were resident peritoneal macrophages. Mouse epidermal cells were isolated by the trypsinization floatation procedure of Yuspa and Harris (1974) using 2-day-old newborn CD- 1 mice. Separated epidermis was minced with scissors, filtered through a nylon mesh to remove clumps, and placed in modified Eagle’s medium. Keratinocytes (4 X IO’) were added to 10 ml of 50% Percoll and centrifuged for 30 min at 15,000 rpm (Fischer et al., 1982). The top 2 ml of the gradient containing flattened squamous cells was discarded and the remaining cells were resuspended in HBSS. Measurement of chemiluminescence responses. Chemiluminescence responses were monitored with an ambient-temperature liquid scintillation counter (Packard Model 3003) operated in the out-of-coincidence mode (Kensler and Trush, 1981). Experiments were begun by incubating 1 X 10’ PMNs in 3 ml HBSS for 10 min

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at 37°C in dark-adapted polyethylene vials. After the background chemiluminescence of each vial was determined, the polycyclic aromatic hydrocarbons (3 PM) or vehicle (dimethyl sulfoxide, 0.1%) was added, any response was noted, and the reactions subsequently were initiated by the addition of TPA (100 rig/ml). Chemiluminescence was monitored for 0.2 min at 75-set intervals, and vials were maintained at 37°C between counting. All additions to the vials as well as the chemiluminescence counting procedure were performed in a darkened room. Results are expressed as counts per unit time minus background. Covalent binding to epidermal DNA. PMNs (1 X 10’) and mouse keratinocytes (3 X 10’) were isolated as described above and cocultured in 3 ml HBSS plus 0.1% glucose for 60 min in the presence of TPA (I 00 @ml) and BP 7,8-dihydrodiol (3 pM) as indicated. Following incubation, cells were centrifuged at 15Og for 10 min, resuspended in 1 ml HBSS, and recentrifuged in 50% Perco11to separate PMNs, which were retained at the top of the gradient, from keratinocytes, which were preselected (sup@ to sediment near the bottom of the gradient. DNA from the keratinocytes was then isolated and covalently bound carcinogen quantified as described below. For the in vivo binding experiments [3H]BP (200 nmol) or [3H]BP trans-7,8-dihydrodiol (400 nmol) was applied to the backs of preshaved mice in a darkened room and the mice were kept in the dark until termination 3 hr later. Animals were also treated with acetone or TPA (17 nmol) at the indicated times. Dorsal skins were treated with a chemical depilatory (Nair) and epidermal DNA was isolated according to the technique of Alexandrov et al. (1983). Epidermal layers from two animals were pooled for each data point. DNA was dissolved in 0.1 mM sodium phosphate, pH 7.0, and DNA content was assayed by the diphenylamine procedure (Giles and Meyers, 1965). Binding of tritiated polycyclic aromatic hydrocarbon equivalents to DNA was quantified by scintillation spectrometry.

RESULTS Recruitment of PMNs into the Skin by Phorbol Esters The accumulation of PMNs is a characteristic feature of the early stages of cutaneous inflammation elicited by tumor promoters and the quantity of PMN influx into the skin may be taken as a measure of the intensity of this disease process (Bach and Goerttler, 197 1; Gschwendt et al., 1984). Measurement of myeloperoxidase, an abundant neutrophil enzyme, offers a simple biochemical ap-

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HOURS

POST TPA APPLICATION

FIG. I. Time course for the accumulation of myleoperoxidase activity in mouse skin following topical application of TPA. Mice were killed at the indicated times following application of 17 nmol TPA and pelts were removed. A 22-mm diameter skin punch was homogenized and assayed for myeloperoxidase activity as described under Methods. Values represent the means k SE of determinations made on groups of four mice at each time point.

preach for estimating PMN content in the skin (Bradley et al., 1982). Figure 1 depicts the time course for the accumulation of myeloperoxidase following topical application of 17 nmol TPA to the skin of CD-l mice. A 5-fold increase in PMN content is observed within 4 hr of TPA application. This influx continues to a maximal 15fold increase that persists from 12 to 20 hr followed by a diminution to near control levels by 48-72 hr. Presented in Fig. 2 are light micrographs of mouse skin treated with either acetone (Fig. 2A) or 17 nmol TPA (Figs. 2B and 2C) 16 hr prior to termination. In accord with the biochemical findings, a substantial influx of PMNs into the dermis can be observed in the TPA-treated skins. Among the phorbol esters, TPA is one of the most active in tumor promotion. Table 1 compares tumor promoters of varying activities with their ability to recruit PMNs into the skin. Phorbol and phorbol triacetate, which are inactive as tumor promoters, are also inactive as stimulators of this PMN response. Phorbol diacetate, phorbol dibutyrate, phorbol dibenzoate, and phorbol didecanoate, which have weak to strong activities as tumor

ET AL.

promoters, show comparable activities as stimulators of PMN recruitment. Retinoyl phorbol acetate, which is a stage II tumor promoter in NMRI mice (Furstenberger et al., 198 l), but a complete promoter in both CD-l and SENCAR mice (Egner et al., 1987; Fischer et al., 1985) was the most effective analog. A similar structure-activity relationship has been observed with this series of phorbol esters for the activation of human PMNs to elaborate reactive oxygen species and a chemiluminescence response (Kensler and Trush, 198 1). Activation PMNs

of BP 7,8-Dihydrodiol

by Murine

Various oxidants have been shown to oxygenate BP 7,8-dihydrodiol. For example, the oxidation of BP 7,8-dihydrodiol by singlet oxygen results in a 9, IO-dioxetane intermediate which decomposes with the emission of photons as indicated by chemiluminescence. Demonstrated in Fig. 3, the addition of 3 PM BP 7,8-dihydrodiol to TPA-stimulated murine PMNs results in a substantial enhancement of chemiluminescence. The chemiluminescence response peaked by 2.5 min following activation of the PMNs with TPA and then gradually dissipated. By contrast, the addition of the parent polycyclic aromatic hydrocarbon BP yielded an insignificant increase in chemiluminescence. No chemiluminescence was observed when either 3 pM BP or BP 7,8-dihydrodiol was admixed with PMNs not stimulated by TPA (data not shown). This differential activation corresponds to the respective responses of BP 7,8dihydrodiol and BP with singlet oxygen-generating systems (Seliger et al., 1982). Comparable responses have also been observed with TPA-activated human PMNs (Trush et al., 1985). However, when phorbol ester-stimulated alveolar or peritoneal macrophages are used as a source of oxidants, no chemiluminescence response is generated with BP 7,8dihydrodiol, implying a central role for the

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TABLE 1

TABLE 2

STRUCTURE-FUNCTION STUDIES ON THE ACTIVITY OF TUMOR PROMOTERS AND ANALOGS TO STIMULATE PMN RECRUITMENT INTO MOUSE SKIN

INFLUENCE OF STIMULATED PMNs ON THE COVABINDING OF BP ~&DIHYDRODIOL TO DNA OF COCULTURED KERATINOCYTES

Compound ~ Retinoylphorbol acetate Phorbol didecanoate Phorbol dibutyrate Phorbol dibenzoate Phorbol triacetate Phorbol diacetate 4-O-methyl TPA a-Phorbol

Induction of myeloperoxidase activity (% of TPA control)” 115+ 15 98-c 11 272 4 9* 3 2+ 1 If 1 Irt 1 No activity

’ Mice were treated with 10 pg of compound and myeloperoxidase activity was assayed in a 22-mm skin punch 16 hr after application. Values represent means + SE of determinations made on four mice. TPA-treated mice had a myeloperoxidase activity of 1.80 +- 0.20 units/punch and acetone-treated mice 0.09 f 0.02 units/ punch.

neutrophil enzyme, myeloperoxidase, in this activation process (data not shown). In addition to activating BP 7,&dihydrodiol to a chemiluminescent product, PMNs can also activate this proximate carcinogen to

Additions Keratinocytes + TPA Keratinocytes + TPA + PMNs

Covalent binding to DNA” 10.2 + 1.86 21.2 + 2.8’

0 Picomoles of tritiated BP 7&dihydrodiol bound per milligram of keratinocyte DNA. b Mean + SEM of five incubations. ’ Differs from control, p < 0.0 1 by Student’s t test.

a DNA-alkylating species. Shown in Table 2, the addition of TPA-stimulated PMNs to murine keratinocytes increases by twofold the covalent binding of BP 7,8-dihydrodiol to the DNA of the epidermal cells compared to those cells exposed to TPA alone in the absence of PMNs. As a consequence, it would appear that either the PMN-derived oxidant and/or the electrophilic derivative of BP 7,8dihydrodiol is a diffusible species that can transverse the plasma membrane and intracellular space of the keratinocyte to reach its nuclear target. Efects of Inj’lammation on the Covalent Binding of BP and BP 7,8-Dihydrodiol to Epidermal DNA

7.0,

.

LENT

6.0.. 6.0.. SP 7,6-dIhyfJdrodk4 4.0.. 3.02.~1.0.. Oi 0

5

10

16

MINUTES

FIG. 3. Time course for the chemiluminescence response elicited from BP or BP 7,8-dihydrodiol in the presence of TPA-stimulated PMNs. Incubations were conducted as described under Methods. (m) TPA (100 rig/ml) plus 3 PM BP 7,8dihydrodiol; (A) TPA plus 3 PM BP; (0) TPA plus DMSO (0.1%) vehicle. Representative curves are displayed.

The effects of a TPA-induced inflammatory response in mouse skin on the covalent binding of BP and BP 7,8-dihydrodiol in vivo are presented in Table 3. Application of 400 nmol of BP 7,8-dihydrodiol to mouse skin produced a level of carcinogen-DNA binding of 25.7 pmol/mg DNA after 3 hr. This level of adduction was not appreciably affected by concurrent treatment with TPA. Recruitment of PMNs into the skin by treatment with TPA 16 hr prior to BP 7,8-dihydrodiol application only marginally increased binding. However, if these recruited PMNs were given a second treatment with TPA at the

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TABLE 3 ROLE OF PMN RECRUITMENT IN THE METABOLIC ACTIVATION OF POLYCYCLICAROMATIC HYDROCARBONSIN MOUSE SKIN Covalent binding of [3H]-labeled carcinogen to epidermal DNA (pmol bound/mg DNA)’

Treatment protocol I

Group

II

Acetone TPA Acetone Acetone TPA TPA

Acetone/BP TPAf BP Acetone/BP 7,8-dihydrodiol TPAJBP 7,8dihydrodiol Acetone/BP 7,8-dihydrodiol TPA/BP 7,8-dihydrodiol

-16

0

10.8 k 0.6 (5) 10.1 If: 0.7 (5) 25.7 f 3.8 (9) 25.2 + 8.2 (5) 30.1 + 4.6 (5) 39.1 f ll.O(9)b

3

Hours 1 Termination a Mean + SE (N). b Differs from acetone-treated control (Group 3), p < 0.05. The data were subjected to a one-way analysis of variance with comparisons among groups performed using the Neuman-Keuls procedure.

time of BP 7,8-dihydrodiol dosing, presumably serving to activate reactive oxygen production and myeloperoxidase release, then a 50% increase in the binding of BP 7,8-dihydrodiol to epidermal DNA was observed. No enhancement of DNA binding of BP was observed with the dual applications of TPA compared to acetone-treated controls. DISCUSSION Reactive oxygen has been implicated in the multiple stages of chemical carcinogenesis (Kensler and Trush, 1985; Kensler and Taffe, 1986) and interest is expanding on the role of reactive oxygen-dependent reactions mediated by PMNs in the pathogenesis of cancer (Roman-France, 1982; Troll and Weiser, 1985). In response to a stimulus such as the phorbol diester TPA, PMNs demonstrate an augmented capacity to generate superoxide anion and hydrogen peroxide-oxygen metabolites whose reactivities are amplified by the catalytic actions of myeloperoxidase.

While PMN-derived oxidants are important to the bactericidal activity of these cells, they are also directly capable of altering biomolecules in other cells as evidenced by PMN-elicited genetic lesions in bacteria and mammalian cells (Weitzman and Stossel, 1981; Weitberg et al., 1983). Additionally, work from our laboratories as well as from others has demonstrated that activated PMNs can participate in the metabolic activation of proximate carcinogens to genotoxic species (Trush et al., 1985, 1986; Tsuruta et al., 1985). Human PMNs stimulated with TPA mediate the activation of BP 7,8-dihydrodiol to species that covalently bind to DNA, elicit mutagenesis in S. typhimurium, and induce sister chromatid exchanges in cocultured V79 fibroblasts. Typically, the activation of carcinogens, such as BP, is catalyzed by enzymes localized to the endoplasmic reticulum and nuclear membranes of the target cell (Gelboin, 1980). Nevertheless, metabolites of BP, including BP 7,8-dihydrodiol, can exit cells and undergo secondary activation elsewhere (Hsu et al., 1978). However, the bio-

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logical relevance of the PMN-mediated activation mechanism in vivo has not hitherto been directly examined. Human malignancies often develop at sites of ongoing inflammation and in many instances inflammation may be a requisite step in the development of cancer at a particular site (Demopoulos et al., 1983; RomanFrance, 1982). The role of inflammation in these situations is undefined, although it has been postulated that free-radical mechanisms may be involved. In the present studies we have taken advantage of the inflammatory properties of epidermal tumor promoters to develop a model system for investigating the influence of an inflammatory state on the metabolic activation of carcinogens in viva Application of TPA leads to an enormous increase in the influx of PMNs into the skin between 12 and 20 hr after exposure (Figs. 1 and 2). Activation of the recruited PMNs by a second application of TPA followed immediately by treatment with the penultimate carcinogen BP 7,8-dihydrodiol results in a 50% increase in the amount of carcinogen irreversibly associated with epidermal DNA as compared to those mice treated with either a single application of TPA or only the acetone vehicle (Table 3). The contrasting situation with BP, in which the double application of TPA had no effect, would mitigate against the involvement of other possible effects of TPA such as increased DNA synthesis and cell proliferation. The dichotomy of effects with BP versus BP 7,8-dihydrodiol in mouse skin is also consistent with the differential abilities of these two compounds to serve as substrates for metabolic activation by stimulated murine PMNs (Fig. 3). Experiments conducted several decades ago examined the influence of irritation induced by croton oil, of which TPA is an active principle, on skin tumor production in mice before a single application of several different carcinogens (Pound, 1968). Consistent with our findings on the lack of an effect of TPA pretreatment on the covalent binding of BP to epidermal DNA (Table 3), pretreat-

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ment with croton oil 24 hr prior to application of BP resulted in only a very modest increase in tumors when tumors were subsequently elicited by multiple applications of croton oil after initiation. Comparable experiments using multiple pretreatments of TPA to recruit and stimulate inflammatory cells prior to initiation and subsequent promotion have not to our knowledge been conducted, but, based on our findings, might be anticipated to exhibit a higher tumor yield if a penultimate initiating agent such as BP 7,8-dihydrodiol were to be used. Although this type of dosing protocol is not directly relevant to the standard initiation-promotion skin carcinogenesis protocols in which promoter application only follows initiation, it might be directly relevant to the process of tumor progression. Hennings et al. (1983) have suggested that a second initiation-like event may facilitate the progression to carcinomas of benign papillomas produced during initiation and promotion in mouse skin. Initiation-promotion protocols typically yield a very low incidence of carcinomas relative to papillomas; however, if papilloma-bearing mice are treated with a second round of an initiating agent, then the progression of the benign tumors to malignant neoplasms is greatly accelerated. Thus, tumors developing at sites of ongoing inflammation may be particularly susceptible to tumor progression. Interestingly, free-radical-generating compounds, such as organic peroxides, are also very effective tumor progressors (O’Connell et al., 1986), suggesting that oxidants generated by activated neutrophils might directly participate in this process in addition to their indirect role in the metabolic activation of DNA-damaging chemicals. Thus, under conditions where there is an accumulation of metabolically stimulated PMNs, such as exists at sites of inflammation, it is conceivable that PMNs could participate either directly or indirectly in the activation of carcinogens and the induction of genetic damage. Further investigations are in progress to assess the importance of this process

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in vivo in target tissues more relevant to major sites of human malignancies, such as the lung. Collectively, these studies should serve to provide a molecular basis for the observed association between the development of malignancies and sites of ongoing inflammation (Demopoulos et al., 1983) as well as to identify the role of chronic inflammatory states as risk factors for neoplasia. ACKNOWLEDGMENTS This work was supported by grants from the USPHS (CA 36380, ES 03760, and ES 00454) and the American Cancer Society (SIG-3). T.W.K. is recipient of a Research Career Development Award CA 0 1230.

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DEMOPOULOS, H. B., PIETRONIGRO, D. D., AND SELIGMANN, M. L. (1983). The development of secondary pathology with free radical reactions as a threshold mechanism. J. Amer. Coil. Toxicol. 2, 173-184. DIX, T. A., AND MARNE~, L. J. (1983). Metabolism of polycyclic aromatic hydrocarbon derivatives to ultimate carcinogens during lipid peroxidation. Science 221,77-79.

EGNER, P. A., TAFFE, B. G., AND KENSLER, T. W. (1987). Effects ofcopper complexes on multistage carcinogenesis. In Biology of Copper Complexes (J. R. J. Sorenson, Ed.), Humana Press, Clifton, NJ, in press. ELING, T., CURTIS. J., BATTISTA, J., AND MARNETT, L. J. (1986). Oxidation of (+)-7,8-dihydroxy-7.8dihydrobenzo[a]pyrene by mouse keratinocytes: Evidence for peroxyl radical- and monooxygenase-dependent metabolism. Curcinogenesis 7, 1957-1963. FISCHER, S. M., HARDIN, L., KLEIN-SZANTO, A., AND SLAGA, T. J. (1985). Retinoyl-phorbol-acetate is a complete promoter in SENCAR mice. Cancer Lett. 27,323-327.

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tumor initiators and unaffected by tumour promoters. Nature {London) 304,6?-69. Hsu,

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