Document M46E9GKBNNkK2GbxQ2wE5eLrk

INTERFERON: PROPERTIES AND CLINICAL USES A. Khan, N.O. Hill, and G.L. Dorn, eds. pp. 589-598, VJadley Institutes of Molecular Medicine, Dallas, IX, 1980. 509 ' INHIBITION OF INTERFERON INDUCTION AS A SCREEN FOR THE CARCINOGENIC POTENTIAL OF CHEMICALS* Gerald Sonnenfeld, Mary Carol Barnes, Julia Schooler and Uldis N. Streips Department of Microbiology and Immunology University of Louisville School of Medicine Louisville, Kentucky, USA ABSTRACT The induction of murine Interferon by Newcastle disease virus has been shown to be inhibited by pre-treatment of fibroblasts with Various carcinogens. The present study has extended the range of carcinogens to include chloroactetaide-- hyde. Chloroethanol and chloroacetic acid, rarely carcinogen ic analogs of chloroace'taldehycfe, had no significant effect on interferon induction by Newcastle disease virus. When polyriboinosinic-polyribocytidylie acid was used as an interferon inducer, induction of interferon was also inhibited by pre treatment of the cells with chloroacetaldebyde. Addition of reduced glutathione, which can trap active metabolites of carcinogens, abrogated the effect of a carcinogen on inter feron induction, suggesting that fibroblasts can activate carcinogens in the inhibition of interferon induction system. Following further verification of the ability of the Inhibit ion of interferon induction system to discriminate among chemicals on the basis of carcinogenic potential, this test could become part of a comprehensive battery of tests for chemical-carcinogenicity. 16 * *' *This work was supported by grants from the American Cancer Society No. INlilC and the Manufacturing Chemists' Associat ion, and the National Aeronautics and Space Administration under Interchange NCA2-ORA00-901. M.C.B. was the recipient of a Graduate Student Research Grant from the University of Louisville Graduate School, and J.S. was a Summer Research Scholar of the University of Louisville School of Dentistry. CMA. 052975 590 INTERFERON: PROPERTIES AND CLINICAL USES INTRODUCTION Many different tests have been developed over the past several years to determine the carcinogenic potential of chemicals. Several of the early tests, such as the Ames Salmonella test (1), vere able to determine the mutagenic potential of chemicals, hut could not consistently discrim inate between mutagens and carcinogens. Several recently de veloped assays, which involve determination of the activat ion of *'S0S" repair in bacteria, have apparently been able to differentiate between carcinogens and mutagens (2,3). How ever, these tests suffer from the drawback that they are carried out in prokaryotic cell systems which may not he analagous to the systems operative following exposure of a mammalian cell to a chemical. Mammalian tests to determine the carcinogenic potential of chemicals have been developed, but also suffer from sev eral drawbacks. Transformation of cell cultures or induct ion of tumors in **<mai models require extended periods of time for the carcinogenic potential of a chemical to be ex pressed. In addition, if a chemical is carcinogenic only at a very low rate, massive quantities of animals would be ren quired to observe the carcinogenic event (4). DeMaeyer and DeMaeyer-Gulgnard have shown in early studies that pre-treatment of rat fibroblasts with several carcinogens Including henzo-( a)-pyrene and 3-methylcholan-- threne resulted in reduced interferon production when the cultures were challenged with vaccinia virus (5,6). Benzo(c)-pyrene, a rarely carcinogenic mutagen and an analog of benzo-(oQ-pyrene, had no effect,on Interferon Induction. Ue have recently extended these studies to a murine system and have shown that several other suspected carcinogens, includ ing Aflatoxin-B,, 2-aminofluorene, styrene oxide, and methyl methanesulfonate, all inhibited interferon Induction by New castle disease virus (NDV) in mouse embryo fibroblasts (7). Ethyl methanesulfonate, a rarely carcinogenic analog of methyl methanesulfonate, also had no effect on interferon induction. The present study represents an extension of the exam ination of the effects of carcinogens on the induction of interferon. Pre-treatment of mouse embryo fibroblasts with the postulated carcinogen chloroacetaldchyde resulted .in CMA 052976 inhibition of interferon induction when the cultures were challenged with NDV. Treatment of the cultures with chloroacetic acid and chloroethanol, rarely carcinogenic analogs of chloracetaldehyde, did not significantly affect interferon induction. These data suggest that the inhibition of inter feron induction system may be useful as an assay for the car cinogenic potential of chemicals, following more extensive studies and confirmation of results. In addition, c&Ioroacetaldehyde treatment inhibited interferon induction by polyriboinosinic-polyribocy tidylic acid (poly I:C), suggesting' that carcinogen treatment does not disrupt the interaction of virus with the cell membrane resulting in inhibited inter feron production. Finally, application of reduced gluta thione to the cell cultures in conjunction with the carcino^ gen benzo--(a) -pyrene resulted in abrogation of the effects of the carcinogen an interferon induction. These data suggest that a system exists in fibroblasts which can activate carcinogens in the interferon inhibition system. MATERIALS AMD METHODS Mouse Embryo Fibroblast Cultures: C57B1/6 derived mice were bred and maintained in our laboratory. Fifteen to eighteen day old embryos were surgically removed from pregnant mice, minced, trypsinized in 0.25Z trypsin 1-300 (ICN Pharaeeuti-- cals, Cleveland, OH) and then suspended in minimum essential medium (Grand Island Biological, Grand Island, NY) supple mented with 10% fetal calf serum, penicillin and streptomycin and glutamine. Second or third passage2culutres were used in all experiments and were plated in 23cm tissue culture flasks (Falcon Plastics, Oxnard CA). Chemicals; Chloroacetaldehyde, chloroethanol and chloroacetic acid were generous gifts of Dr. John Wong, Department of Chemistry, University of Louisville. Polyriboinosinlc and polyrlbocytidylic acids were obtained from P-L Biochemicals, Milwaukee, WI. Benzo-(a)-pyrene was obtained from Aldrich Chemical Company, Milwaukee, WI. Reduced Glutathione was obtained from Calbiochem, La Jolla, CA. Interferon Induction: Mouse Type I interferon was produced in fibroblasts with the Herts strain of Newcastle disease virus, as described elsewhere (8). After inactivation of residual inducing virus by pH 2 treatment at A C for four days, the tissue culture supernatants were assayed for anti viral (interferon) activity. Polyrihoinosinic and polyribo- CMA 052977 592 INTERFERON: PROPERTIES AND CLINICAL IISES cytidylic acid were completed to poly I:C by heating at 45C fot one hour. Type I interferon was induced with poly I:C by adding 50 vg of poly X:C to tissue cultures for 90 min., and* adding additional fresh medium to the .mouse embryo fibroblast cultures. DEAE-Dextran was included with' the poly I:C to insure maximum of induction of interferon (9). After twenty-four hours of incubation, the culture supernat ants were harvested and assayed for antiviral activity. Interferon Assay: Interferon titers-were determined by plaque reduction on mouse L-929 cells using the Indiana strain of bovine vesicular stomatitis virus CIO). The inter feron titer corresponded to the reciprocal of the highest dilution of test sample that reduced virus plaques by 50Z. One interferon unit in this assay equals 0.88 NIH-C--002--904-- 511 reference units. RESULTS Chloroacetaldehyde, chloroethanol, and ehloroacetle add were soltiblized in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations in tissue culture medium. The chemicals were then added to different confluent monolayers of mouse embryo fibroblasts. Following twenty-four hours of Incubation at 37C, the culture supernatants were removed and Interferon was Induced with NDV. The results shown In Table 1 indicate that only treatment with chloroacetaldehyde resulted in decreased Interferon production of 50Z or greater. TABLE 1 EFFECT OF PRETREATMENT OF CELL CULTURES WITH CllLOROACETALDEHYDE AND ITS ANALOGS ON INTERFERON INDUCTION BY NDV Treatment* Interferon Titer NDV Only DMSO + NDV Chloroacetaldehyde + NDV Chloroacetic acid + NDV Chloroethanol + NDV 300 500 69 225 209 X Decrease .-- -- 77Z 2SZ 30Z *A11 chemicals were applied at a concentration of 0.005 |M CMA 052978 SECTION IV: IN VITRO TESTING 33 Since the interferon assay has an innate two-fold vari ability due to the biological nature of the assay (11), only differences of 50% or greater were considered to demonstrate an effect of a carcinogen on interferon induction. When chloroacetaldehyde, chloracetic acid and chloro-- ethanol were applied to confluent monolayers and interferon was induced with poly IsC, Inhibition of interferon induction of 50% or greater was again only observed in- chloroacet aldehyde treated cultures (Table 2). Cbloroacetic acid and chloroethanol had much less of an effect on the induction of interferon. TABLE 2 EFFECT OF FKETREATMENT OF CELL CULTURES WITH CHLOROACET ALDEHYDE AND ITS ANALOGS ON INDUCTION OF INTERFERON BY POLY IsC Treatment* Poly I:C only DMSO + poly IsC Chloroacetaldehyde + poly IsC Chloroaeetic acid + poly IsC Chloroethanol + poly IsC Interferon Titer 430 385 187 268 304 Z Decrease -- 10% 57% 37% 29% *A11 chemicals were applied at a concentration of 0.005 pm. Benzo-( a) -pyrene was solublized in DMSO and applied to confluent monolayers of mouse embryo fibroblasts at a con centration of 0.5 pm. As has been previously reported for induction of interferon by NDV (7), this treatment resulted in a significant drop in the titer of interferon induced by poly 1:C (Table 3). Addition of reduced glutathione with the benzo-( o)-pyrene resulted in abrogation, at least in part, of the inhibitory effects of benzo-(c)-pyrene on interferon induction by poly 1:C (Table 3). Glutathione itself had minimal if any effect on the induction of inter feron (Table 3). CMA. 052979 594 INTERFERON: PROPERTIES AND CLINICAL USES TABLE 3 EFFECT OF CONCOMITANT ADDITION OF GLUTATHIONE AND BENZO-(o)-PYRENE ON INTERFERON INDUCTION BY POL.Y ItC Treatment Poly 1:C only Benzo-- (a) --pyrene + poly I:C. Benzo5-(a) -pyrene + 0.1 pm glutathione + poly 1:C Benzo-( b)-pyrene -1- 0.01 pm glutathione + poly X:C 0.1 pm glutathione + poly I:C 0.01 pm glutathione + poly 1:C Interferon Titer 266 76 181 150 347 196 * Z Decrease -- 71Z 31Z * 43Z* 26Z Addition of glutathione to cultures ot mouse embryo fibro blasts did not result in the induction of detectable levels of interferon. DISCUSSION The induction of Type 1 interferon has been shown to be inhibited by several carcinogenic chemicals (3-7). Of par ticular interest is the observation that when several pairs of highly carcinogenic chemicals and their rarely or noncarcinogenic analogs, e.g. benxo-(o)-pyrene and benzo-(c)pyrene, ethyl methanesulfonate and methyl'methanesulfonate were tested, only application of the proven carcinogen resulted in the inhibition of interferon induction (5-7). Ve have now extended this observation to include the presumed carcinogen chloroacetaldehyde, and its rarely carcinogenic analogs chloro acetic acid and chloroethanol. After many additional pairs of carcinogens and analogs are tested in the future, the inhibition of interferon induction by chemicals may prove useful in the screening of chemicals for carcino genic potential. I CMA. 052980 szction iv: in vrrno izsTinc 525 Dimethyl sulfoxide has previously been shown to have a r.cn-statistically significant minimal effect on the induction of interferon by NDV (7). In the present study, no effect of DMSO on interferon induction was observed. The mechanism of the inhibition of interferon induction was also examined in the present study. Previous work has shown that pre-treatment of rat fibroblasts with carcinogens did not inhibit the plaqulng efficiency of vaccinia virus (5). Since poly I:C induction of interferon is also 'inhibited by pre-treatment of fibroblasts with carcinogens. It is not likely that the -carcinogen treatment is affecting the binding of viruses to the cell membrane or the budding of viruses. Many carcinogens must be activated by microsomal oxidases in order to form final active products before an effect in bacterial or mammalian systems can be observed (12). Benzo-- (a)-pyrene is included among these chemicals. Reduced glutathione can trap these active products and prevent the occurence of a carcinogenic event (12). Application of reduced glutathione to the inhibition of interferon induction system resulted in at least partial abrogation of the effects of benzo--(a)--pyrene on Interferon Induction. These data suggest that fibroblasts must contain the type of activation system for carcinogens that renders them potent for the inhibition of Interferon induction. REFERENCES 1. Ames, B.N., Durstan, U.E., and Yamasaki, E. Carcinogens* are muCagens: A simple test system combining liver homogenates for activation and bacteria for assay. Proc. Natl. Acad. Sci.. 70:2281-2285, 1973. 2. Moreau, D., Balione, A., and Devoret R. Prophage lambda Induction in Escherichia coli K12 envA envB: A highly sensitive test for potential carcinogens. Froc. Natl. Acad. Sci.. 73:3700-3704, 1976. 3. Streips, U.N., Laumbach, A.D., and Yasbin, R.E. Bacillus subtilus assays for mutation and DNA repair. In, Bacterial Mutation Monitors for Active Metabolites of Chemical Carcinogens: ed.. Felkner, I.C. Harccl Dekkcr, New York, New York. In Press, 1979. CMA. 052981 596 INTERFERON: PROPERTIES AND CLINICAL USES 4. Ames, B.N. Identifying environmental chemicals causing mutations and cancer. Science. 204:587--593. 1979. 5. PeMaeyer-Guignard, J., and DeMaeyer, E. Effect of car cinogenic and noncarcinogenic hydrocarbons on interferon synthesis and virus plaque development. J. Natl, Cancer Inst.. 34:265-276, 1965. 6. DeMaeyer, E., and De-Maeyer-Guignard, J. ' Inhibition by 3-methylcholanthrene of interferon formation in rat embryo cells infected with Sindbis virus. . J. Natl, Cancer Inst.. 32:1313-1321, 1964. 7. Barnes, M.C., Streips, U.N.,* and Sonnenfeld, G. Inhibit ion of interferon induction: Differentiates among chemi cals on the basis of carcinogenic potential. Submitted for publication, 1979. 8. Sonnenfeld, G., Mandel, A.D., and Merlgan, T.C. The immunosuppressive effect of Type II mouse Interferon on antibody production. Cell. Immunol.. 34:193-206^ 1977.* 9. Dianzlnl, F., Can'tagalll, Gagnoni, S., and Rita, G. Effect of DEAE-dextran on production of interferon by . synthetic double-stranded ENA in L cell cultures. Froc. Soc. Exp. Biol. Med.. 128:708-711. 1968. 10. Brodeur, B.R., Weinstein, 7., Melmon, K.L., and Merlgan, T.C. Keclprocal changes in interferon production and Immune responses of mouse spleen cells fractionated over columns of lnsolubli2cd conjugates of histamine. Cell. Immunol.. 29:363-372.. 1977. 11. Lockhart, R.Z. Criteria for acceptance of a viral inhibitor as an interferon and a general description of the biological properties of known interferons. In, Interferon and Interferon Inducers; ed,, Finter, N.B. American Elsevier, Mew 7ork, Mew York, pp 11-28, 1973. 12. Chasseaud, L.F. Glutathione (reduced) and glutathione S-transferase blocks the carcinogenic event by trapping the ultimate metabolites. Adv. Cancer Res.. 29:176-255. 1979. `` CMA 052982 SECTION IV: IN VITRO TESTING 597 . D--S i.vil UNKNOWN: Do you think that this effect is restricted only to these groups of carcinogens and works also with aflatexins? SONNENFELD: It does work with aflatoxin. We have shown it with aflatoxin previously as well and there has just been a report published using aflatoxins and the B1 aflatoxin. UNKNOWN: And what about tumor promotors? SONNENFELD: There have been reports using coal dust and also asbestos fibers as well, that `there was reduced induc tion of interferon. The alfatoxin story is very interesting because these investigators at Western Virginia University used four different types of aflatoxin. One type being more potent carcinogen than the other and they found that the de gree of inhibition of induction was related to the potency of the type of aflatoxin that they used. DEGRE: So you have any idea whether other gall types could be used for the same tests? SONNENFELD: We have used fibroblast. DeMeyer has used fibroblasts. There has been one study in vitro. I cannot remember the chemical right off hand, but if mice were treated with this chemical they could not produce in vivo serum Inter* feron. I do not know about other things. Of course, we would like to get into studies with Type II interferon as well in lymphoid cells but to this point this type of work has only been done using fibroblast. GROB: You might have mentioned it but could you please repeat what the pre treatment schedule was before interferon induction. SONNENFELD: What we would do was treat those cells with the carcinogens for twenty-four hours and then wash and make sure that there was no toxicity of the carcinogen. UNKNOWN: Jay,, just to support you, the emulsifiers that we used severely reduced the ability of cells to pro duce interferon. If you use them in very small concentra tions in leukocyte culture, they cause chromosome breaks to a substantial degree higher than the normal controls. CMA 052983 598 INTERFERON: PROPERTIES AND CLINICAL USES SONNEKFELD: Which might suggest some carcinogenic potential of the material. UNKNOWN: Yes, I couldn11 agree more on the need of shifting from a prokaryotic system to an eukaryotic system and 1 think that your approach is particularly'exciting and promising, especially considering that you can use a human . cell. * . . SONNENFELD: Yes, we have begun studies along those lines. , * CMA 052984