Evaluation of in vitro reporter gene induction assays for use in a rapid prescreen for compound selection to test specificity in the Tg.AC mouse short-term carcinogenicity assay

Under ICH guidelines, short-term carcinogenicity assays such as the Tg,AC a ssay are allowed alternatives for one species in the 2-year rodent bioassay . The Tg.AC transgenic mouse, which carries the v-Ha-ras oncogene under con trol of the zeta -globin promoter, develops skin papillomas in response to dermal application of carcinogens and tumor promoters. The appropriate spec ificity of the Tg,AC model for testing pharmaceuticals has not been systema tically evaluated, The selection of candidate test compounds among noncarci nogenic pharmaceuticals would be aided by a high-throughput in vitro prescr een correlative of activity in the in vivo Tg.AC assay, Here we describe th e development of a prescreen based on correct response to 24 compounds test ed previously in Tg,AC mice. The in vitro prescreens, chosen to reflect mol ecular pathways possibly involved in Tg.AC papilloma formation, consisted o f a zeta -globin promoter-luciferase construct stably expressed in K562 cel ls (Zeta-Luc) and three of the stress-response element-chloramphenicol acet yltransferase (CAT) fusion constructs stably expressed in HepG2 cells that are part of the CAT-Tox (L)iver assay, The stress response elements chosen were the c-fos promoter, the gadd153 promoter, and p53 response element rep eats. Of the four assays, the gadd153-CAT assay showed the strongest concor dance with activity in the Tg.AC assay, correctly classifying 78% of Tg.AC positive and 83% of Tg.AC negative compounds. The correlation was further i mproved by adding the Zeta-Luc assay as a second-stage screen. These cell-b ased assays will be used in a novel approach to selecting candidate compoun ds that challenge the specificity of the Tg,AC assay toward pharmaceuticals .