An in vitro assay of chemical mutagenicity in rat mammary epithelial and fibroblast cells
Epithelial cells are the primary site for carcinogenesis in most tissues, while stromal cells rarely develop into tumours. To investigate the basis of this differential susceptibility, three cell lines were developed from the oral epithelium (BBR/OE), mammary epithelium (BBR/ME), and stromal mammary fibroblasts (BBR/MFib) of transgenc Big Blue rats. These animals carry multiple chromosomal copies of a bacteriophage transgene for mutant frequency analysis using a selective assay. The different cell types were characterized cytogenetically and tested for chemically-induced cytotoxicity and for genotoxicity in the transgene ('cII'). The metabolic basis for their relative responses was investigated. For the direct-acting mutagen 'N'-ethyl-' N'-nitrosourea, all cell lines were equally sensitive to mutagenesis in the 'cII' gene but the fibroblast cell line showed greater resistance to cytotoxicity. For the dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-' b']pyridine (PhIP), rat liver S9 was used to supply cytochrome P450 and conjugating enzymes. Mutagenicity was observed in the fibroblast cells, but the greater toxicity of PhIP to the epithelial cells prevented a definitive evaluation of mutagenicity. When the cells were treated with N-OH-PbIP, the epithelial cells were again more sensitive to cytotoxicity but a small increase in mutagenicity was observed for both mammary cell lines. RT-PCR analysis showed that cytochrome P450 enzymes 1A1 and 1B1 are expressed at low levels in both cell lines, with greater 1B1 expression seen in the fibroblast cells. In contrast, greater 'O'-acetyltransferase expression was measured in the mammary epithelial cells. It can be concluded that insufficient P450 activity is expressed to produce the hydroxylated metabolite of PhIP and that other conjugating enzymes may play a role in producing the ultimate mutagen. For the combustion products benzo['a']pyrene (B['a']P) and dimethyl-benz['a']anthracene (DMBA), a strong cytotoxicity and mutagenicity response was observed only in the fibroblast cells. Furthermore, this response occurred in the absence of exogenous bioactivation and could be reduced, in a dose-dependent fashion, by the P450 1A1 and 1B1 inhibitor [alpha]-naphthoflavone. Continued work with these cell lines may provide further insight into the role that chemical mutagens play in carcinogenesis in specific cell populations.