A method for the amplification of chemically induced transformation in C3H/10T1/2 clone 8 cells: its use as a...

A method has been developed by which to amplify expression of phenotypic transformation of C3H/10T1/2 clone 8 mouse embryo cells not otherwise observed in the standard transformation assay. The expression of transformed foci was amplified by subcultivating chemically treated target cells after they had reached confluence and replating them at subconfluent cell densities. Conditions leading to the expression of the highest numbers of transformed foci include a) a cell seeding density for chemical treatment of 1 X 10(4) cells/dish, b) subculture 4 weeks after treatment, and c) replating cells at a density of 2 X 10(5) cells/-dish. Agents capable of inducing transformation in the standard assay (e.g., 4,4'-bis(dimethylamino)benzophenone, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and others) also yielded transformation in the replating assay. The more marginal transforming activities of chemicals such as ethyl methanesulfonate, 7-(bromomethyl)-12-methylbenz[a]anthracene, and N-methyl-N'-nitro-N-nitrosoguanidine were enhanced by the amplification procedure. Compounds that failed to elicit focal transformation in the standard assay (e.g., dibenz[a,h]anthracene, Tris(2,3-dibromopropyl) phosphate, lead acetate, benzidine, propyleneimine, N-hydroxy-2-fluorenylacetamide, and numerous other compounds of various chemical classes) induced significant levels of phenotypic transformation upon amplification. Noncarcinogens (e.g., phenanthrene, anthracene, 2-aminobiphenyl, cycloheximide, and others) failed to cause significant phenotypic transformation even when cells were replated. To further enhance the applicability of this new replating system, an exogenous source of metabolic activation was added: a 9,000 X g supernatant from Aroclor 1254-induced rat hepatic S-9. This activation system was found a) to be only minimally cytotoxic by itself and b) to be able to mediate NADPH-dependent, dose-dependent toxicity, and transformation by activating the procarcinogens dimethylnitrosamine, 2-naphthylamine, 2-aminoanthracene, and aflatoxin B1. With the use of this revised assay, 14 coded and 23 model compounds were tested. Agreement with in vivo results was observed to be over 85%. The marked sensitivity and discriminatory ability of this revised assay procedure suggest its usefulness as a screen for potential carcinogens of diverse chemical structure.