Until relatively recently, a measurement of mutational effects in vivo was rather difficult to per- form. One of the more popular assays utilized in this area was the dominant lethal assay, in which male mice are exposed to a potential genotoxic stimulus and mated with untreated female mice; the percentage of pregnancies or number of implants is then determined (Lockhart et al.,1992). While the method is fairly easy to perform, relatively few carcinogenic agents have been studied by this method. Similarly, the production of sperm abnormalities in mice by the adminis- tration of chemical agents in vivo has not found general use as a short-term mutagenic assay (Wyrobek and Bruce, 1975).
Figure 13.1 Scheme of the Ames test for mutagenesis of chemicals in Salmonella bacterial strains. The upper part of the figure outlines the preparation of the S-9 mixture of enzymes and particulates prepared from rodent liver taken from animals previously administered an agent to induce the concentration of such metabolizing enzymes. The Salmonella, which require histidine for their growth (his–), are grown in the presence of histidine, separated from the growth media, and added with the test chemical and S-9 mix as well as soft agar containing a trace of histidine, which allows the cells to undergo one or two divisions (required for mutation fixation). The S-9 and soft agar mix is transferred to a petri dish while still warm, incubated for several days, and the colonies that develop in the absence of histidine are counted. (Modified from McCann, 1983, with permission of the author and publisher.)
Figure 13.2 Outline of chemically induced mutation in mouse cell lines with thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT) as the target gene. (Reproduced from Pitot and Dragan, 1996, with permission of the authors and publisher.)
In recent years, with a variety of genetic tools available, genetically engineered cells and animals have been developed that have found use in short-term mutagenesis assays. The four examples given in Table 13.3 are those most commonly used for mutational analysis in vivo. The first three involve genetically engineered animals containing transgenes within which are com- ponents of the lac operon of Escherichia coli, a set of coordinately regulated genes involved in lactose metabolism. A schematic representation of the lac operon is seen in Figure 13.3. Some details of the function of the lac operon are given in the figure legend. Basically, the lacI and lacZ genes are the ones utilized in the mutational assays. As noted in the figure, mutations in the lacI gene will alter the regulation of expression of the lacZ gene, which codes for β-galactosi- dase activity. Thus, the transgene contains either one or the other of the operons. Mutations in the bacterial transgene are determined by the methods seen in Figure 13.4. In this technique,
Figure 13.3 Schematic representation of the lac operon in E. coli. A. The lacI gene codes for protein that forms a homotetramer that binds to the lacO operator sequence. Binding of the repressor to lacO pre- vents transcription of lacZ. B. Transcription of lacZ occurs in the presence of the inducing agent, isopropyl- β-thiogalactoside (IPTG). Mutations of the lacI may result in partial or complete inactivation of the lac repressor, the lacI tetrameric protein. Furthermore, mutations in the lacZ gene may prevent interaction with the repressor or may be nonfunctional, resulting in no production of the structural gene, lacZ. (Reproduced from Provost et al., 1993, with permission of the authors and publisher.)
DNA is extracted from the tissue of interest, and because of the nature of the transgene, con- struct may be packaged into a bacterial virus, lambda, which then infects the bacteria, E. coli, on a lawn of bacterial growth on a dish as noted in the figure. By selecting appropriate bacterial strains and media, one can isolate mutant phage and analyze the sequence of the lacI or lacZ gene as appropriate. Thus, one may obtain both the number of mutations per unit DNA from the mouse or, more importantly, the actual sequence changes induced by the mutagenic action of the original agent. The rpsL transgene works by a similar mechanism but by a different metabolic pathway (Gondo et al., 1996).
Since several of the transgenic animals are commercially patented, this assay may entail some expense, but it is relatively versatile for an in vivo assay for mutagenic identification. However, its ability to detect nonmutagenic carcinogens is doubtful. Of interest is the fact that with at least one carcinogenic agent, ethylnitrosourea, the relative sensitivities of mutations in- duced in the lacI transgene and hprt, an endogenous gene, were essentially identical (Skopek et al., 1995). Species differences occur with different carcinogens in that, for example, aflatoxin B1 treatment resulted in a much greater number of mutations in the lacI rat than in the lacI mouse (Dycaico et al., 1996). External ionizing radiation was not very mutagenic in the lacZ transgenic mouse (Takahashi et al., 1998), but of interest is the finding that the promoting agent phenobar- bital enhanced mutation frequency in the livers of lacZ transgenic mice treated with diethylnitro- samine (Okada et al., 1997). While a significant number of spontaneous mutations occur in the
Figure 13.4 Sequence of steps utilized in the determination of the mutagenicity of chemicals in trans- genic rodent mutagenicity assays in vivo. The details of the test are briefly discussed in the text or the reader may refer to the original article. (From Recio, 1995; reproduced with permission of the author and publisher.)
transgene, as yet this does not appear to be an insurmountable problem (de Boer et al., 1998). Thus, the potential for utilizing such transgenic models for the in vivo assay of mutagenesis is clearly bright. However, their effectiveness in identifying promoting and progressor agents has yet to be validated.