[CANCER RESEARCH 43,1945-1950, May 1983] 0008-5472/83/0043-OOOOS02.00 Chemical Enhancement of Viral Transformation in Syrian Hamster Embryo Cells by Gaseous and Volatile Chlorinated Methanes and Ethanes1 George G. Hatch,2 Patricia D. Mamay, Mark L. Ayer, Bruce C. Casto, and Stephen Nesnow Northrop Services, Inc., Research Triangle Park, North Carolina 27709 [G. G. H., P. D. M., M. L A., B. C. C.], and Carcinogenesis and Metabolism Branch, Genetic Toxicology Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711 [S. N.] ABSTRACT Methods were developed for exposing cells in vitro to gases or vapors of volatilized organic liquids. Compounds were se lected for their industrial importance, environmental impact, and suspected role in the etiology of some human cancers. Exposure chambers were designed for easy insertion of dishes of cultured cells and were equipped with inlet and outlet ports for introduc tion and purging of test gases. A gas delivery system utilizing a mass flow meter was used for the quantitative distribution of test gases into exposure chambers. For volatile compounds, appropriate volumes of cold (4°)liquids in glass Petri dishes were quickly placed into chambers, the system sealed, and the compounds rapidly volatilized at 37°. For exposure, the cells and chambers were placed in an incubator and rocked at a constant rate so that a portion of the cells was always in direct contact with the test gases or vapors. Known sample volumes were removed after various treatment times and test gas con centrations determined by standard gas Chromatographie tech niques. After exposure, the cells were removed and assayed for viability and increased sensitivity to viral transformation. Under these experimental conditions, the volatile liquids 1,1,1-trichlo- roethane, dichloromethane, chloroform, 1,2-dichloroethane, and 1,1-dichloroethane significantly enhanced transformation of Syr ian hamster embryo cells by SA7 adenovirus, while acetone exerted no effect. The gases chloromethane and vinyl chloride were also active in this test system, while bromomethane, meth ane, and ethane were inactive. Incorporation of some of these compounds into liquid cell culture medium for cell treatment was either unsuccessful or produced only a weak enhancement re sponse. Methodology is now available to evaluate volatile and gaseous carcinogens or mutagens and can be used to identify their mechanisms of action and the relative hazards of these agents to human health. INTRODUCTION To prevent human cancers caused by exposure to environ mental carcinogens, suitable methodologies and bioassays must be developed that will permit the detection of all classes and physical forms of carcinogenic agents. Quantitative cell culture assay systems provide procedures for detecting potential carcin- 'This work was supported by the United States Environmental Protection Agency under Contract No. 68-02-2566. A preliminary account of this work was presented at the Symposium on Genotoxic Effects of Airborne Agents, Brookhaven, N. Y., February 1981 (13). This paper has been reviewed by the Health Effects Research Laboratory, United States Environmental Protection Agency, and ap proved for publication. Mention of commercial products does not constitute en dorsement or recommendation for use. 2To whom requests for reprints should be addressed. Received September 16,1982; accepted January 14,1983. MAY 1983 ogens in a relatively short period of time compared to in vivo tests. Mammalian cells in culture are particularly appropriate for such assays because they represent an extension of the in vivo experimental models, eliminate host factors, afford a means to control test chemical levels, and can be conducted in a controlled environment. Quantitative linear dose-response relationships have been demonstrated for cell transformation assays using fibroblasts from hamster embryo cells (1, 2, 8, 9). The transfor mation event data are consistent with a one-hit phenomenon indicating a direct cause-and-effect relationship (7, 8). A short-term in vitro assay for carcinogens and mutagens has been developed (2,4, 5, 8,13,15) that determines the ability of various chemical carcinogens to enhance the adenovirus trans formation of SHE3 cells. The viral enhancement assay reflects the capacity of a chemical to damage cellular DNA either directly or indirectly and provides a rapid mammalian cell bioassay sys tem for screening suspected genotoxic environmental chemicals. This bioassay has detected activity for a large number of com pounds from a variety of chemical classes, including alcohols and phenols, aliphatic amines, alkyl sulfates and sultanes, aro matic amines, aryl halides, carbohydrates and derivatives, hy- droxylamines, mycotoxins, polycyclic hydrocarbons, hydrazines, metal salts, steroid hormones, and chlorinated hydrocarbons (2, 3). The last 4 classes are generally negative in standard bacterial (Salmonella) assays (21, 22). Data from approximately 105 test performances in 29 different chemical classes have been pub lished for the enhancement of viral transformation assay (2, 3). With that assay as an index of carcinogenicity, 94% of 130 chemicals with known positive or negative activity agreed with their current in vivo classifications regarding carcinogenicity. Volatile or gaseous chlorinated compounds are ubiquitous environmental contaminants. Their use in industrial workplaces is widespread; for example, the annual production of 1,2-dichlo roethane is 11.8 billion pounds/year in the United States (17). Small amounts of chloroform and related chemicals can be detected in municipal drinking water supplies (23), and DCM is used in certain food extraction processes (17). Previous attempts to evaluate the genotoxic potential of TCE and 1,2-dichloroethane by viral enhancement assays with incor poration into liquid cell culture media were unsuccessful or produced only weak or inconsistent enhancement responses. This report documents the successful development of methods for treating mammalian cell cultures with volatile organic liquids and gaseous compounds and measuring the chemical enhance ment of DNA viral transformation in hamster embryo cells. The compounds studied were: TCE, DCM, 1,2-dichloroethane, 1,1- 3 The abbreviations used are: SHE, Syrian hamster embryo; TCE, 1,1,1 -trichto- roethane: DCM, dichloromethane; MDM, modified Dulbecco's medium; FBS. fetal bovine serum; TF, transformation frequency. This One 1945 DWJC-958-74GG on May 1, 2016. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from