The procedure for the induction of DMBA-induced mammary lesions has been described in detail previously. BALB/c female mice obtained are used in the study. The animals are pretreated subcutaneously with 1 microgram 17beta-estradiol + 1 mg progesterone for 9 days. The thoracic mammary glands are excised and incubated in Waymouth's MB752 medium with 5 micrograms insulin, 5 micrograms prolactin, 1 microgram aldosterone and 1 microgram hydrocortisone per mil medium for 10 days with a 24 hour exposure of the glands to DMBA (2 micrograms/ml) on day 3. The glands are then cultured for 14 days in the presence of 5 micrograms/ml insulin alone. The other experimental conditions include 37 degrees C temperature, 50% 02, 5% CO2, and 45% N2 atmosphere. Two control groups are included in each test, a DMSO and a DMBA Control. The chemopreventive agents are included in the medium for the first 10 days of culture at 5 different concentrations ranging from 1 x 10(-9)M to 1 x 10(-5)M. At the end of 24 day experiment, the glands are fixed in formalin, stained with alum carmine and evaluated for the incidence and multiplicity of the mammary lesions in these glands. There are 36 chemopreventive agents to be evaluated by this assay.
