The procedure for the induction of DMBA-induced mammary lesions has been described in detail previously (Telang et al., PNAS 76:5886, 1979; Mehta and Moon, Cancer Res. 46:5832, 1986). BALB/c female mice obtained are used in the study. The animals are pretreated subcutaneously with 1 g 17 -estradiol + 1 mg progesterone for 9 days. The thoracic mammary glands are excised and incubated in Waymouth's MB752 medium with 5 g insulin, 5 g prolactin, 1 g aldosterone and 1 g hydrocortisone per ml medium for 10 days with a 24 hour exposure of the glands to DMBA (2 g/ml) on day 3. The glands are then cultured for 14 days in the presence of 5 g/ml insulin alone. The other experimental conditions include 37xC temperature, 50% O2, 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 10-9M to 1 x 10-5M. 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 25 chemopreventive agents to be evaluated by this assay.
