[CANCER RESEARCH 41, 4341-4345, November 1981] 0008-5472 /81 /0041 -OOOOS02.00 Reduction of Tumorigenicity and of Dihydrodiol Formation by Fluorine Substitution in the Angular Rings of Dibenzo(a,/)pyrene1 Stephen S. Hecht,2 Edmond J. LaVoie, Victoria Bedenko, Linda Pingaro, Shoichi Katayama, Dietrich Hoffmann, Dennis J. Sardella, Eliahu Boger, and Roland E. Lehr Nay/or Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595 [S. S. H., E. J. L. V. B., L P.. S. K., D. H.); Chemistry Department, Boston College, Chestnut Hill, Massachusetts 02167 [D. J. S., E. B.]; and Chemistry Department, University of Oklahoma, Norman, Oklahoma 73019 [R.E.L.] ABSTRACT The tumor-initiating activities on mouse skin and in vitro metabolism of dibenzo(a,/)pyrene, 2-fluorodibenzo(a,/)pyrene, 3-fluorodibenzo(a,/)pyrene, and 2,10-difluorodibenzo(a,/)py- rene were compared. After an initiating dose of 500 jug, fol lowed by promotion with tetradecanoylphorbol acetate, di- benzo(a,/)pyrene induced skin tumors in 85% of the mice and caused 5.8 skin tumors/mouse. The corresponding tumori- genie activities for the fluorinated compounds were: 2-fluoro- dibenzo(a,/)pyrene (85%; 1.7 tumors/mouse); 3-fluorodi- benzo(a,/)pyrene (80%; 3.1 tumors/mouse); and 2,10-difluo- rodibenzo(a,/)pyrene (10%; 0.1 tumors/mouse). After an initi ating dose of 100 /ig, only dibenzo(a,/)pyrene showed signif icant tumor-initiating activity. 3,4-Dihydro-3,4-dihydroxydi- benzo(a,/)pyrene was identified as a metabolite of dibenzo(a,0pyrene formed by the 9000 x g supernatant from the livers of Aroclor 1254-pretreated rats. Another dihydrodiol was tentatively identified as 1,2-dihydro-1,2-dihydroxydiben- zo(a,/)pyrene. The formation of these angular ring dihydrodiols was inhibited in the metabolism of 2-fluorodiben- zo(a,/)pyrene and 3-fluorodibenzo(a,/)pyrene. Angular ring dihydrodiols were not detected in the metabolism of 2,10-di- fluorodibenzo(a,0pyrene. These results suggest that an angular ring dihydrodiol, 3,4-dihydro-3,4-dihydroxydi- benzo(a,0pyrene, which can form a bay-region dihydrodiol epoxide, may be a proximate carcinogen of dibenzo(a,/)pyrene. INTRODUCTION DB(a,i)P3 (see Chart 1) is one of the most potent sarcoma- genie agents known (5, 12). Its sarcomagenic activity exceeds that of benzo(a)pyrene. DB(a,i)P is also a tumor initiator and complete carcinogen on mouse skin, with activity somewhat lower than benzo(a)pyrene (10). However, little is known about the metabolic activation of the dibenzopyrenes despite their presence in coal tar, polluted urban air, and the carcinogenic neutral fractions of cigarette smoke (4,11,17, 21 ). Relatively few studies on the metabolism of DB(a,i)P have 1 This study was supported by the National Institute of Environmental Health Sciences Grant ES 02030 and National Cancer Institute Grants CA 23454 and 22985. Presented in part at the Fifth International Symposium on Polynuclear Aromatic Hydrocarbons, Battello Columbus Laboratories, October, 1980 (7). 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: DB(a,i)P, dibenzo(a,i)pyrene; 2-F-DB(a,i)P, 2- fluorodibenzo(a,/)pyrene; 3-F-DB(a,i)P, 3-fluorodibenzo(a,/)pyrene; 2,10-diF- DB(a,i)P, 2,10-difluorodibenzo(a,/)pyrene; 3,4-dihydrc-3.4-dihydroxyDB(a,i)P, 3,4-dihydro-3,4-dihydroxydibenzo(a,/)pyrene; HPLC, high pressure liquid chro- matography; TCPO, 1,1,1-trichloropropene oxide; 1,2-dihydro-1,2-dihydrox- yDB(a,i)P, 1,2-dihydro-1,2-dihydroxydibenzo(a,Opyrene. Received March 16. 1981 ; accepted July 30, 1981. been reported. Waterfall and Sims (24) described the formation of dihydrodiols and phenols as metabolites of DB(a,i)P in vitro, as well as the enzymatically mediated binding of DB(a,i)P to cellular macromolecules. In earlier in vivo studies in mice, the rate of removal of DB(a,i)P from the site of injection, as well as its excretion in urine and feces, was investigated (13, 15, 23). In the present study, we have used the fluorine probe meth odology to investigate the metabolic activation of DB(a,i)P. This approach has been used in previous studies on carcinogen activation (8, 14, 18, 19). We have compared the tumor-initi ating activity and in vitro metabolism of DB(a,i)P and 3 of its angular ring-fluorinated derivatives, 2-F-DB(a,i)P, 3-F-DB(a,i)P, and 2,10-diF-DB(a,i)P (see Chart 1). The results indicate that 3,4-dihydro-3,4-dihydroxyDB(a,i)P may be a proximate carcin ogen of DB(a,i)P. MATERIALS AND METHODS Apparatus. HPLC was performed with a Waters Associates Model ALC/GPC-204 high-speed liquid Chromatograph equipped with a Model 6000A solvent delivery system, a Model U6K septumless injec tor, a Model 440 UV/visible detector, a Varian Fluorichrom fluores cence detector (excitation maximum of 360 nm with emission measured from 430 to 610 nm), and a 4.6- x 250-mm Hibar II Lichrosorb RP-18 10-m column (EM Reagents, Cincinnati, Ohio). Chromatographie sep arations of metabolites were carried out using a solvent program as follows: 50% methanol in H2O for 20 min and then linear to 100% methanol in 40 min at a flow rate of 2 ml/min and a system pressure of 2000 to 4000 psi. UV spectra were obtained on a Gary Model 118 spectrometer, and fluorescence spectra were determined with an American Instrument Co. Spectrophosphorimeter. Chemicals. DB(a,i)P was obtained from ICN K & K Life Sciences Group, Plainview. N. Y. 2-F-DB(a,i)P, 3-F-DB(a,i)P, 2,10-diF-DB(a,i)P, and 3,4-dihydro-3,4-dihydroxyDB(a,i)P were synthesized; spectral and analytical data are given in Refs. 2, 16, and 20. Prior to bioassay, the dibenzopyrenes were assayed by HPLC (isocratic; methanol) using fluorescence detection. No impurities greater than 1% were detected. However, subsequent analysis with UV detection at 254 nm did reveal the presence of an impurity in the samples of DB(a,i)P, 3-F-DB(a,i)P, and 2,lO-diF-DB(a,i)P. In the case of DB(a,i)P, it was identified by its UV spectrum (23), by its mass spectrum, and by conversion to DB(a,i)P as 6,7-dihydrodibenzo(a,/)pyrene. It eluted prior to DB(a,i)P on HPLC and was present to the extent of 5% in the DB(a,i)P used in the bioassays. A compound with similar UV and relative retention volume and presumably a similar structure was also detected in the samples of 3-F-DB(a,i)P (8%) and 2,10-diF-DB(a,i)P (3%). All impurities were removed by HPLC prior to metabolic studies. 3,4-Dihydro-3,4- dihydroxyDB(a,i)P was also purified by HPLC using the Chromato graphie conditions used for separations of metabolites prior to its use as a reference standard. No impurities were detected in 2-F-DB(a,i)P. NADP* and glucose-6-phosphate were obtained from Sigma Chemical Co., St. Louis, Mo. TCPO was obtained from Aldrich Chemical Co., NOVEMBER 1981 4341 Research. on September 14, 2021. © 1981 American Association for Cancer cancerres.aacrjournals.org Downloaded from