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Oncology JS: 98-10! (1981) j Effect of Carcinogens and Analogs on Interferon Induction Mary Carol Barnes, Uldis N. Streips and Gerald Sonnenfeld Department of Microbiology and Immunology, University of Louisville, School of Medicine, Louisville. JCy. j Key W rds. Interferon - Carcinogen inhibition t ; Abstract. Pretreatment of mouse embryo fibroblasts with several chemicals, including 7,12-dimethylbenz-(a)| anthracene, 2-aminofluorcne, aflatoxin Bi, bcnzo-{a)-pyrene, styrene oxide, and the No. 4 fraction of tobacco < smoke condensate, resulted in severely reduced production of interferon when the cells were challenged with Newcastle disease virus. All of the above chemicals are proven or strongly suggested carcinogens. When the analogs methyl methanesulfonate, a potent carcinogen, and ethyl methanesulfonate, a weak carcinogen, were applied to cells, interferon induction was only inhibited by the methyl methanesulfonate. Therefore, carcinogens may directly inhibit the induction of interferon by Newcastle disease virus. Introduction interferon is now being used in several clinical trials for the treatment of various types of tumors, including non-Hodgkin's-type lymphomas [1]. Therefore, it is of interest to determine the type of interactions that exist between interferon and tumor-inducing substances, such as carcinogens. Early experiments by DeMaeyer and DeMaeyerGuignard (2, 3} suggested that application of known chemical carcinogens, such as benzo-(a)-pyrene, to rat embryo fibroblast cultures prior to challenge with vac cinia virus resulted in inhibition of the induction of interferon by the virus. An additional finding of sig nificance was that bcnzo-(e)-pyrenc, a weak carcino gen, had no effect on the induction of interferon. These data suggested that carcinogens might have a specific inhibitory effect on the induction of interferon. The results of the present study confirm and extend these observations. Several known and suspected po tent carcinogens, including methyl methanesulfonate (MMS), markedly inhibited the induction of interferon by Newcastle disease virus (NDV) in mouse embryo fibroblasts. The rarely or weakly carcinogenic analog of |mMS, ethyl methanesulfonate (EMS) [4, 5] had little or no effect on the induction of interferon by NDV. Therefore, the new data suggest that interferon induc tion is inhibited by pretreatment of cell cultures with an extended group of potent carcinogens. Materials and Methods Mouse Embryo Fibroblast Cultures. B6 H-2k Iyt2.1 mice, a gift of Thomas Huff and Samuel Welthausen, Department of Microbiology and Immunology, University of Louisville. Ky,, were bred and main tained in our laboratory. 15- to 18-day-old embryos were surgically removed from mothers, minced, trypsinized in 0.25% trypsin 1-300 (Pharmaceuticals, Cleveland. Ohio) and then suspended in Gibco minimal essential medium (MEM) with 10% fetal calf serum. Second or third passage cultures were used in all experiments and were plated in Falcon 25 cm1 tissue culture flasks, and were used immediately upon reaching confluency. Chemicals. Aflatoxin 8,, 2-aminofluorene, bcnzo-(ct )-pyrcne, EMS and MMS were obtained from Aldrich Chemical Co.. Milwaukee. Wise. 7.12-Dimclhylbcnz-(u)-anthraccnc and No. 4 fraction of to bacco smoke condensate were received from the Kentucky Tobacco Health and Research Institute. Lexington. Ky. Dimethylsulfoxide (DMSO) was obtained from J.T. Baker Chemical Co.. I'hillipshurg. N.J. Styrene oxide was kindly presided by Dr. John H'ong. Depart ment of Chemistry, University of Louisville, Ky. Interferon Production. Mouse type 1 interferon was produced in fibroblasts with the Herts strain of NDV. as described elsewhere (6). Carcinogens and Interferon 99 R&S 116569 After inactivation of residual inducing vims by pH 2 treatment at 4 *C for 4 days, the tissue culture supernatant Quid was assayed for interferon activity. Interferon Assay. Interferon titers were determined by plaque reduction on mouse L-92U cells using Indiana strain bovine vesicular stomatitis virus. The interferon titer corresponded to the reciprocal of the highest dilution Of test sample that reduced virus plaques by JO. One interferon unit equals 0.88 NIH HG-002-904-511 reference units (6). Results Table 1. Effect of carcinogens on interferon production Treatment of fibroblasts Newcastle disease virus 7,12-Dimethyibenz-(a)anthracene 4- NDV 39.0 fiM 3.9/rAf 2-Aminofluotene + NDV 0.050 pW 0.003 nM Antiviral titer 1.000 % decrease from control -- 100 90 86 91 21 98 247 81 Viability of Mouse Embryo Fibroblast Cultures Mouse embryo fibroblasts were allowed to grow into a confluent monolayer. After reaching confluency, dif ferent cultures were treated for 24 h with several con centrations of the chemicals used in this study. These chemicals included 7,12-dimethylbenz-(a)-anthracene, 2-amin fluorene, aflatoxin Bt, No. 4 fraction of tobac co smoke condensate, benzo-(a)-pyrene, MMS, EMS, DMSO and ethanol. Immediately after removal of the chemicals, the fibroblast cultures were stained with 2 x neutral red, a vital stain. All of the cultures that were treated with the dosages of chemicals that were used in the study were >95% viable by staining criteria. Addi tional cultures that had chemical-containing medium replaced with fresh MEM remained viable for at least another 24 h. Effect of Known Carcinogens on Interferon Induction The known carcinogens 2-aminofluorenc and 7,12dimcthylbenz-(a)-anthracene were diluted in MEM and added to confluent fibroblast cultures. After 24 h of incubation, the culture supernatants were removed. The cultures were then challenged with NDV for 24 h and the supernatants were harvested, pH 2 treated, and then assayed for interferon activity. Reductions in inter feron titers 90% were obtained when compared to non carcinogen treated controls (table 1). Several different dosages of chemicals were initially used to determine the dosage for optimum effect without affecting via bility of the cultures, and the data in table I represent the optimal dosages. Effects of Several Chemicals on Interferon Induction Mouse embryo fibroblasts were treated with several different dosages of various chemicals before attempted induction of interferon with NDV. Aflatoxin Bt, No. 4 fraction of tobacco smoke condensate, benzo-(a)-pyrene. and styrene oxide treatments all inhibited the induction of interferon by 90% or greater (table II). Table IL Blind study of the effect of various chemicals on inter feron induction Chemical treatment' Interferon titer SE % decrease Pnov1 from control None (NDV control) Aflatoxin B| 0.05 pM No. 4 fraction, tobacco smoke1 condensate, lOx Benzo-a-pyrene, 0.03 /tM Styrene oxide, 0.05 pM 1.456 455 105 82 89 20 122 74 14960 -- 93 95 92 90 _ <0.05 <0.05 <0.05 <0.05 1 Mouse embryo fibroblasts wen pretreated with the appropriate chemical for 24 h. Supernatants were removed, cells washed and NDV applied for 24 b. Cell supernatants were then harvested and assayed for antiviral activity. 1 Statistical analysis was performed by means of a Student's T analysis. 1 Arbitrary dilutions of the No. 4 fraction of tobacco smoke con densate were used. Table lit, Blind study of the effect of a weak and a potent car cinogen on interferon production Chemical treatment' Interferon titer SE % decrease1 P from control None (NDV control) Ethyl methancsulfonate 0.05 aM Methyl methanesulfonate 0.05 nM 1,456 455 t,680 294 137 82 -- 91 _ >0.5 <0.05 ' Experimental protocol as in tabic 11. 1 EMS and MMS were dissolved in ethanol only. Table II represents the effects of optimum dosages of the chemicals used. DMSO was used as a solvent for all of the above chemicals. Pretreatment of fibroblast cultures with DMSO resulted in a small (0--40%), not 100 Barnes/Streips/Soiinenfcld statistically significant decrease in the titers of inter feron induced by NDV. EMS and MMS were solubilized in ethanol. Pre treatment of mouse fibroblasts with ethanol had no effect on the titers of interferon induced by NDV. Pretreatment of the cell cultures with EMS had a minimal effect on the induction of interferon by NDV, while MMS pretreatment severely decreased the titer of inter feron induced by NDV (table 111). Discussion * The results of the present study indicate that several well-known carcinogens, including 7,12-dimethylbenz(a)-anthracene, benzo-(a)-pyrene, 2-aminofluorene, aflatoxin Blt and the No. 4 fraction of tobacco smoke condensate, all inhibited the induction of interferon by NDV. Styrene oxide, an important industrial chemical, which was positive in the Ames assay, but negative in all Bacillus assays done to date, also inhibited the in duction of interferon by NDV. Further studies of the carcinogenic potential of styrene oxide may be required. DMSO has been used in clinical trials as a drug for the treatment of arthritis. However, these trials have been suspended due to the teratogenic potential of the chemical [7, 8], It is of interest to note that pretreat ment of cells with DMSO did not result in the signifi cant inhibition of interferon induction by NDV. EMS and MMS are closely related analogs which differ with respect to carcinogenic potential as deter mined by tumor induction (4, 5]. MMS is a highly car cinogenic mutagen, while EMS is a rare or weakly car cinogenic mutagen. In the present study, MMS strongly inhibited the induction of interferon by NDV, while EMS had no significant effect. In a recent study, fibro blasts were pretreated with four different forms of aflatoxin [9]. The degree of inhibition of interferon in duction correlated with the proven carcinogenic poten tial of the aflatoxin forms, i.e. the more carcinogenic the form of aflatoxin, the greater the inhibition of inter feron induction. In view of these findings and the pre viously reported discrimination between benzo-(a)pytene and benzo-(c)-pyrene [3] and the presently observed discrimination between EMS and MMS, car cinogens may directly inhibit the induction of inter feron. The mechanism by which carcinogens inhibit the in duction of interferon is not known. A toxic effect of the carcinogens to the cell cultures does not seem likely. since the results of this study indicate that the targeytk cells are alive when they fail to produce interfcronVr However, a general effect of the carcinogens on DNA, RNA, or protein synthesis and differential dosage ef- ,, fects of potent and weak carcinogens cannot be ruled out and are the subject of future studies. :j Other possible mechanisms include alteration of the cell membrane by the carcinogen resulting in reduced interaction of virus with the cells. Recent studies show- ~- ing that pretreatment of cells with carcinogens inhibited ^ the induction of interferon by poly-inosinic-poly-cyti- ' dylic acid, a nonviral inducer of interferon [10], In view *; of those studies and the earlier finding that carcinogen pretreatment does not inhibit the replication of the ; viruses used to induce interferon [2], it is unlikely that . virus-membrane interactions are severely disrupted by carcinogen treatment of cells. 'fj Another possible mechanism could involve induction _ of a protease by the carcinogen in a manner analogous to bacterial `SOS* repair systems [11]. This protease could degrade induced interferon, or interfere with the control mechanisms for interferon induction. The true nature of the proposed effect of carcinogens on inter- ; feron induction is the subject of extended studies. . '4 The implications of these studies toward an under standing of the mechanisms of carcinogenesis are obscure. It is possible to speculate that there may bW balance between interferon levels and oncogenesis and that balance may be upset by the action of a carcino gen. In addition, if further studies continue to indicate a differentia] effect on interferon induction by strong and weak carcinogens, the effect of a chemical on inter feron induction may have some use in determination of the carcinogenic potential of that chemical. : Acknowledgements Wc thank Dr. R.E. Yasbin of the Pennsylvania State University for providing unpublished data and helpful suggestions. This work was supported by grams from the Manufacturing Chemists' Associa tion, the American Cancer Society No. IN-11IC. and the National Aeronautics and Space Administration under Interchange NCA2OR400-V01. M.C. B. was the recipient of a Graduate Student Re* search Grant from the Graduate School of the University of Louis ville. References 1 Mcrigan, T.C.; Sikora, K.; Breeden, J.H.; Levy, R., and Rose** berg. S. A.: Preliminary observations on the effect of human leukc R&S 116570 | farcmogens and Inicrteron I interferon in non-Hodgltin's lymphoma. New F.ngl. J. Med. ^^99; 1449-1453 (1979). 2 DeMacycr. E. and DeMacyer-Guignard, J.: Inhibition by 3-methyl; cholanthrcne of interferon formation in rat embryo cells infected with Sindbis virus. J. natn. Cancer Inst. 32: 1317-1321 (1964). ! 3 DeMacyer-Guignard. J. and DoMueyer. E.: fitted of carcinogenic and noncarcinogcnie hydrocarbons on interferon synthesis and virus plaque development. J. natn. Cancer Inst. 34; 265-276 (1965). 4 Roc. F. L.C.; Mitchley. B.C. V,, and Wallets. M.: Tests for car cinogenesis using newborn mice: 1.2-benzanthracene, 2-naphthylamine, 2-naphthylhydroxylamine, and ethyl methanesulfonate. Br. J. Cancer 77: 255-260(1963). 5 Casio, B.C.; Janosko, N.. and DiPaolo, J.A.: Development of a focus assay for transformation of hamster cells in vitro by chemical carcinogens. Cancer Res. 37: 35011-3515 (1977). 6 Sonnenfeld. G.; Mandcl. A.D.. and Merigan. T.C.: The immuno suppressive effect of type 11 mouse interferon on antibody pro duction. Cell. Immunol. 34; 193-206(1977). 7 Caujollc, F. M. E.: Caujolle, D. H.: Cros.S. B., and Calvct. M.-M. J.: Limits of toxic and teratogenic tolerance of dimethylsulfoxide. Ann. N. Y. Acad. Sci. 141: 110-126 (1967). 101 S Rubin, L.F.: Toxicity of dimethylsulfoxide, alone and in combina tion. Ann. N.Y. Acad. Sci.243: 98-103 (1975). 9 Hahon. N.; Booth. J.A., and Stewart, J.D.: Aflatoxin inhibition ot viral interferon induction. Antimicrob. Agents Chemothcr. 16: 277 (1979). 10 Sonnenfeld, G.; Barnes. M.C.; Schooler, J.. and Strvipv. U.N.: Inhibition of interferon induction as a screen for the carcinogenic potential of chemicals; in Khan. Hill, Dorn. Interferon: properties and clinical uses, pp. 589-598 (Wadlcy Inst. Molec. Med-, Dallas 1980). 11 Witkin, E.M.: Ultraviolet mutagenesis and inducible DNA repair in Escherichia eoli. Bad. Rev. 40: 869-907 (1976).* Dr. Gerald Sonnenfeld, Department of Microbiology and Immunology, Univenity of Louisville School of Medicine. Health Sciences Center. Louisville, KY 40292 (USA) DO CO 07 CJI -1 i '