(CANCER RESEARCH 46, 2863-2865, June l986J studies have used the two-stage tumorigenesis protocol, concen trating on events during initiation and promotion. Since carci noma development is relatively rare (approximately 10% of papillomas progress to carcinomas) and carcinoma is a late event in the two-stage protocol, little has been reported on the modification of malignant progression. The observation that carcinomas occur relatively frequently in models in which car cinogens are applied repetitively has led to the proposal that progression is closely related to initiation (6). Several investi gators have treated mouse skin papillomas with tumor initiators and observed increased carcinoma formation compared to con trol mice treated with the potent tumor promoter 12-O-tetra decanoylphorbol-13-acetate (7, 8). These researcherssuggested that progression to malignancy was the result ofgenetic changes causedby mutagenic agents. Benzoyl peroxide is a free radical generating agent, used as an industrial polymerizing agent, as a curing agent for flour and cheeseand asan additive in cosmetics and pharmaceuticals, including those used in the treatment of acne (9, 10). It is moderately active as a tumor promoter but is inactive as a complete carcinogen or tumor initiator (1 1, 12). Similar to other tumor promoters, benzoyl peroxide induces an increase in dark basal keratinocytes, epidermal hyperplasia, terminal differentiation, and ODC levels, and it inhibits intercellular communication in both mouse (12, 13) and human cells (14, 15). As it is true for other agents, these promotional effects may be related to benzoyl peroxide's free-radical generating ability (12). In previous work (16) tumor promotion with ben zoyl peroxide (20 mg twice weekly) was found to cause an unexpectedly high rate of carcinomas relative to papillomas. We hypothesized that in addition to its promotional activity benzoyl peroxide may be highly active in malignant progression, the third stage of carcinogenesis. This report shows that some tumor promoters have the ability to alter malignant progres sion. MATERIALS AND METHODS Female SENCAR mice were receivedat 5—7wk of age (Harlan Laboratories, Indianapolis, IN). Skin tumors were chemically induced using a standard two-stage initiation-promotion protocol. Initiation was accomplished by a single topical application of 10 nmol DMBA4 (Aldrich, Milwaukee, WI) on the shaveddorsal skin. Biweekly promo tion with 1 ;zgof 12-O-tetradecanoylphorbol-l3-acetate (L. C. Services Corp., Worburn, MA) was begun 2 wk after initiation. All solutions were applied in 0.2 ml of acetone. Beginning with wk 21, one group of papilloma-bearing mice (21 animals) continued to receive twice-weekly applications ofTPA (1 gig), while a second group (20 animals) was begun on twice-weeklytreat ments of 20 mg of benzoyl peroxide. Treatments were continued until theterminationof theexperimentat wk 40. Papilloma yield between groups was expressed as the number of papillomaspermouse±SEandcomparedbyStudent'st test.Carci nogenesis was followed by recording the cumulative number of mice whichdevelopedcarcinomasandthecumulativenumberofcarcinomas 4 The abbreviations used are: DMBA, 7,12-dimethylbenzanthri O-tetradecanoylphorbol-13-acetate; GGT,-y-glutamyltransferase. ABSTRACF Chemical carcinogenesis in mouse skin can be divided into the proc esses ofinitiation, promotion, and progression. The free-radical generator benzoyl peroxide is moderately active during the promotion stage. Re petitive treatment ofmouse benign skin tumors (papillomas) with benzoyl peroxide (20 mg, twice weekly) increased the number of cumulative carcinomas per group by 325% and the number of keratoacanthomas by 44% compared to tumor-bearing Sencar mice treated with the promoter 12-O-tetradecunoylphorbol-13-acetate. The lack of increase in the num her ofcumulative papillomas per group due to benzoylperoxidetreatment suggests that benzoyl peroxide enhanced the progression of preexisting papillomas. The ability of benzoyl peroxide to enhance the progression of benign tumors to cancer should be considered when determining the human risk from exposure to this widely used chemical agent in addition, biological assays specifically testing malignant progression may be es sential and beneficial for determining an agent's carcinogenic risk. INTRODUCFION Carcinogenesis in several experimental systems can be di vided into processes of initiation, promotion, progression, and metastasis. The separation between initiation and promotion has been clearly demonstrated in mouse skin (1) in which a single subthreshold dose of a carcinogen (initiation) followed by repetitive applications of a noncarcinogenic, nonmutagenic agent (promotion) produces first papillomas and then squamous cell carcinomas (two-stage tumorigenesis). Tumors that arise from repetitive applications of a carcinogen (complete carci nogenesis) are theoretically the result of both the initiating and promoting abilities of most carcinogens. Evidence indicates that initiation involves the induction of permanent genetic alterations and is essentially irreversible. Most, but not all, initiators are classified as mutagens in stand ard in vitro mutagenesis assays (2). Promotion seems to involve clonal expansion of initiated cells leading to the expression of benign tumors. In mouse skin, promotion can be subdivided into two stages: an early stage that correlates with dark basal keratinocyte induction and a late stage that correlates with epidermal hyperplasia and ornithine decarboxylase induction (3, 4). The biological significance of many of the biochemical and molecular events during promotion is not clear. Progres sion, the process by which a benign tumor develops malignant characteristics is proposed to involve a number of genetic alterations. In addition to their invasive behavior, mouse skin squamous cell carcinomas appear morphologically and geneti cally more deviant than either normal or benign tissue (5). Squamous cell carcinomas of mouse skin rarely metastasize to other organs, but their metastasis can be increased by applica tions of alkylating agents.3 During the last two decadesmost mouse skin carcinogenesis Received 11/4/85; revised 2/10/86; accepted 2/25/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I These studies were supported by NIH grants CA-34890, CA-34962, CA 34521,andCA 38863. 2 To whom requests for reprints should be addressed. 3 1. J. Slaga, unpublished results. TPA, 12- 2863 Enhanced Malignant Progression of Mouse Skin Tumors by the Free@Radical Generator Benzoyl Peroxide' John F. O'Connell,2 Andres J. P. Klein-Szanto, Donna M. DiGiovanni, JoAnn W. Fries, and Thomas J. Slaga The University ofTexas System Cancer Center, SciencePark-ResearchDivision, Smithvile, Texas 78957 Research. on January 22, 2022. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from