Mammalian cell transformation systems

Mammalian Cell Transformation Systems as Prescreens for Identifying Carcinogens in the Environment... 

TABLE 1. Mammalian Cell Transformation Systems Employing Fibroblast... 

Positive results from the in vitro mammalian cell transformation test indicate that the test substance produces phenotypic changes in cultured mammalian cells associated with malignant transformation in vivo. None of the in vitro test endpoints has an established mechanistic link with cancer. Some of the test systems are capable of detecting tumor promoters. 

In most instances the specimens will be self-evident (e.g., samples of blood, plasma, serum, urine, spinal fluid, aqueous humor, organs, tissues, and tissue fractions that are taken from a test system with the intention of performing an examination or analysis). In other instances the definition may not be as clear. For example, the assay plates used in the mammalian cell transformation assay and the mammalian point mutation assay are considered specimens even though they bear many of the attributes of a test system. For these assays, the originally plated cells plus media and excipients are the test system. After treatment with the test or... 

This usually means that the system should detect three major classes of mutation—genic, chromosomal, and genomic. Only with additional justification would other end points, such as chemical damage to DMA and mammalian cell transformation (neoplastic transformation), be recommended as part of a mutagenicity test battery. Such systems can be validated by demonstrating that they predict mutation. 

In this series of bioassay experiments, several carcinogens failed to transform certain batches of hamster embryo cells, presumably because of a lack of endogenous enzymes required for the metabolic activation of these compounds. In short-term bacterial and mammalian cell mutagenesis systems, these enzymes are routinely provided by the addition of rat liver homogenates. As mentioned earlier, diethylnitrosamine and urethane, which failed to transform hamster embryo cells directly, were activated in a host-mediated in vivo-in vitro cell transformation assay in which the chemicals were inoculated intraperitoneally. In our laboratory, when A -2-acetylaminofluorene, 4-aminoazobenzene, auramine, diethylnitrosamine, 3-methoxy-4-aminoazoben-zene, Natulan, 2-nitrofluorene, p-rosaniline, and urethane were tested in the presence of hamster liver homogenates and appropriate cofactors, all except 4-aminoazobenzene gave positive results. The liver homogenates were prepared from hamsters that were not treated with enzyme inducers such as Aroclor 1254 or phenobarbital. 

The cell transformation assay uses mammalian cell culture systems to detect phenotypic changes in vitro induced by chemical substances associated with malignant transformation in vivo. Widely used cells include SHE (Isfort et al, 1996), C3H10T1/2 (Mondal and Heidelberger, 1970) and Balb/3T3 (Matthews et al, 1993). 

The genotoxic data are largely negative, although 1,1,1-trichloroethane was mutagenic in some Salrmnella assays and induced chromosomal aberrations in Chinese hamster ovary cells and cell transformation in mammalian systems. ... 

In cultured mammalian cells, acrylonitrile induced DNA strand breakage, gene mutation, sister chromatid exchanges and chromosomal aberrations, but not aneuploidy or unscheduled DNA synthesis in rat hepatocytes, at least if the silver grain counting method was used. [Studies using the less reliable scintillation counting method have not been summarized.] Cell transformation was induced in several test systems and gap-junctional intercellular communication was inhibited in one study with Chinese hamster V79 cells. 

Methyl iodide induces DNA damage and is mutagenic to bacteria in the presence or absence of an exogenous metabolic system. It induces mitotic recombination in yeast and mutations in cultured mammalian cells. It induces transformation in Syrian hamster embryo cells but not in C3H lOT A cells. 

Transfection, DNA uptake in eukaryotic systems, often is more problematic then bacterial transformation the mode of DNA uptake is poorly understood and efficiency is much lower. In yeast, cell walls can be digested with degradative enzymes to yield fragile protoplasts, which are then able to take up DNA. Cell walls are resynthesized after removal of the degrading enzymes. Mammalian cells take up DNA after precipitation onto their surface with calcium phosphate [Fugene 6 (Roche) Lipofectin (Life Technologies) Effectene (Qiagen)]. Electroporation is often more efficient for transfection in eukaryotic cell systems, especially in yeasts. 

Mammalian cells in culture permit several end points to be measured simultaneously in the same cell system. These include malignant transformations, chromosomal changes, and DNA damage and repair, as well as specific-locus mutation.26... 

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