Short-Term Tests-Mutagenesis Assays

29 May

A variety of short-term  tests, almost all of which are involved in direct or indirect assays of mutagenicity,  both in vivo and in vitro, have now been developed  and are used to aid in the identification of potential carcinogens. However, virtually all of these methods are of limited use in directly establishing the estimation of the risk that such chemicals pose for the human popula- tion. As seen in earlier chapters in this text (Chapters 5 and 6), a ubiquitous characteristic  of neoplastic cells is the presence of a variety of different types of mutations. The fact that many but not all carcinogenic agents are mutagenic or may be metabolized to mutagenic forms further establishes the importance of mutations in the development of the neoplastic process. It is on this basis that short-term  tests for mutagenicity  were developed  to identify potential carcinogenic agents on the basis of their capacity for inducing mutations in DNA in cells in vitro or in vivo.

Table 13.3 lists many of the more commonly used short-term tests for mutagenicity and thus carcinogenic  potential. The most widely utilized of these mutagenicity  assays was origi- nally developed in Salmonella typhimurium by Bruce Ames and associates (Ames et al., 1975). In this assay, bacterial cells that are deficient in DNA repair and lack the ability to grow in the

absence of histidine are treated with several dose levels of the test compound, after which rever- sion to the histidine-positive phenotype is ascertained. Because bacteria differ in their metabolic capabilities compared with mammals, a drug-metabolizing system is added to these assays. Spe- cifically, the 9000 g supernatant (S9) that results from centrifuging a liver homogenate prepared from a rat treated with an inducer of multiple P-450s, such as Aroclor 1254, is used in combina- tion with an NADPH regenerating system. The method for performing the Salmonella assay (the Ames assay) is described in Figure 13.1. Several different lines of Salmonella have been gener- ated to permit the detection  of point mutations  (TA100,  TA1535)  and frameshift  mutations (TA98, TA1537, TA1538), and the assay is continuously being refined. Typically, five dose lev- els of the test compound are used in addition to the solvent control. Activation-dependent  and activation-independent  positive control mutagenic  substances  are tested concurrently.  Certain types of carcinogens are not detected by these bacterial mutagenicity assays, including hormonal carcinogens, metals, agents that have a multiple-target-organ  mode of action, and agents with a nongenotoxic  mode of action. This bacterial reverse mutation system, when performed in the presence of a mammalian S9 activation system, is, however, a very sensitive screen for the detec- tion of many mutagenic agents.

In addition to the bacterial mutational assay, several in vitro mammalian cell mutation as- says exist, including  the mouse lymphoma  L5178Y (MOLY)  assay and the Chinese hamster ovary (CHO) assay. These mammalian mutagenicity assays use either the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or the thymidine kinase (TK) gene as the end point. The basis for these assays is seen in Figure 13.2. They are similar to the Ames assay in that the phe- notypic expression  of a mutation in a single-copy  gene is compared  in treated and untreated cells. These assays are frequently  performed  in the presence  of an exogenous  metabolizing source, such as an epithelial cell layer that has been irradiated. The mammalian mutation test systems are forward mutation assays in which the heterozygous state of a gene is used as a tool to detect genetic damage that might result in the loss of a phenotype, e.g., growth in the presence of a toxic compound. In CHO cells, the X-linked HPGRT locus is used as the target gene for analysis. This enzyme is important in purine salvage and allows the incorporation of toxic purine analogs such as 6-thioguanine and 8-azaguanine into DNA, resulting in inhibition of cell growth and/or cell death. Alternatively, a mutation in this gene that results in phenotype loss may permit colony formation in the presence of toxic analogs. Assays based on the forward mutation of TK are similar in that colony formation in the presence of a DNA-damaging  agent is scored in the presence of a pyrimidine analog. Because these short-term tests are based on the premise that carcinogens damage DNA, their concordance with the chronic bioassay in vivo (see below) is only between 30% and 80%. In addition, the results of tests are coincident with each other and tend to detect the same types of carcinogens  without providing  the battery approach that has been suggested. Among the short-term mutagenicity tests that use mutation as the end point, the Ames assay has been the best studied and has been applied to the greatest number of compounds.

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