Detection of Ductal Dysplasia in Mammary Outgrowths Derived from Carcinogen-treated Virgin Female BALB/c Mice1

These studies were undertaken to determine if altered growth potential of mammary epithelial cells could be detected in outgrowths derived from monodispersed mammary cells of virgin female BALB/c mice previously exposed to ionizing radiation or 7,12-dimethylbenz(a)anthracene (DMBA). Monodispersed mammary epithelial cells were obtained by enzymatic dissociation of mammary tissues of 12-week-old virgin female BALB/c mice. Twenty-four hr prior to cell disso ciation, donor animals were exposed to either 100 rads of yray irradiation, 0.25 mg of DMBA, or 0.075 mg of DMBA. Control donors were untreated. Mammary outgrowths were then derived from these donor cells by injecting either 105 or 104 cells into the gland-free mammary fat pads of three-weekold virgin female BALB/c mice. Ten weeks after the injection of cells, the outgrowths were examined and classified. Mam mary outgrowths were classified either as having a normal ductal architecture or as having ductal dysplasia. Ductal dysplasias were further classified on the basis of an index of severity, which was an arbitrary index based on the number of abnormal ductal structures within each lesion. The data indi cated that treatment of donor animals with either y-radiation or DMBA increased the frequency of ductal lesions over control levels; however, both the frequency and severity of the lesions depended on the number of cells which were injected into the fat pad. When outgrowths were derived by injection of 105 cells into the gland-free fat pads, lesion frequencies in outgrowths from control and treated cells were: 3.3%, control; 15.7%, yrays; 5.3%, 0.25 mg DMBA; in these groups only a few severe lesions were detected. In outgrowths derived from 104 cells, less severe lesions (Class I lesions) were common in all groups and occurred in approximately 10 to 15% of the outgrowths. The frequency of severe (Class II and III) ductal dysplasia, however, was increased by treatment in these groups, occur ring in 4.5% of control outgrowths in 15.6, 14.9, and 14.3% of the outgrowths derived from donor cells treated with 100 rads y-rays, 0.075 mg DMBA, and 0.25 mg DMBA, respectively. Thus, these data indicated that ductal dysplasias were more common and more severe in outgrowths derived from 104 rather than 10s cells. The ductal lesions observed in this study resembled both morphologically and histologically ductal ab normalities which have been associated with the pathogenesis of mammary carcinoma in both rats and mice. 1 Research supported jointly by the Environmental Protection Agency under Agreement 40-740-78, and the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation. 2 Supported by Carcinogenesis Training Grant CA 09104 from the NIH. To whom requests for reprints should be addressed. Received September 16, 1981 ; accepted January 20, 1982. INTRODUCTION Mammary tumors which arise in rodents, whether by the action of chemical carcinogens or the MuMTV,3 are usually preceded by discrete lesions which have high neoplastic po tential relative to normal tissue (15, 17). One such lesion is the HAN. HAN can be induced in both rats (3, 27) and mice (9) by administration of chemical carcinogens and by the MuMTV in mice (7). Transplantation studies have indicated that this lesion has tumor potential greater than that of normal tissue in both species, independent of the mode of induction (2, 4, 5, 16, 22). DeOme ef al. (6) have provided evidence which suggests that the expression of nodule-transformed cells as HAN in mice bearing the MuMTV is modified by the presence and architec ture of normal mammary cells. This hypothesis stems from the observation that, when mammary cells from mice with MuMTV expressed were enzymatically dissociated and transplanted into the gland-free fat pads of syngeneic hosts, the expression of HAN was markedly enhanced in the outgrowths which re sulted. Further support for this hypothesis came from Medina era/. (18), who showed that the tumor potential of HAN-derived outgrowth lines could be enhanced by enzymatic dissociation and transplantation of these cells into the gland-free fat pads of virgin hosts. In the rat, the ductal origin of mammary carcinoma has been well established (20, 23, 24), and there is a good deal of evidence which indicates that these tumors are preceded by ductal dysplasias (10, 14, 21). Mammary lesions of ductal origin have also been observed in mice with MuMTV unex pressed following treatment with DMBA, and although in the past less emphasis has been placed on the importance of these lesions in mouse mammary tumorigenesis, evidence is avail able which indicates that these lesions also have high neoplas tic potential (17). Recent reports have suggested that the cell dissociation assay can be used to detect ductal mammary lesions induced by exposure of mice with MuMTV unexpressed to DMBA (8, 11) or to ionizing radiation (8). In this report, we present the results of our experiments which indicated that altered growth potential of mammary epi thelial cells induced by exposure of donor mice to chemical and physical carcinogens resulted in the expression of mam mary ductal dysplasia which could be detected using the cell dissociation technique. In addition, we report evidence which indicated that the expression of ductal dysplasias was influ enced by the number of donor cells which were transplanted into the host fat pads. 3 The abbreviations used are: MuMTV, murine mammary tumor virus; HAN, hyperplastic alveolar nodule(s): DMBA, 7,12-dimethylbenz(a)anthracene.