TOWARD AN UNDERSTANDING OF THE USE OF TRANSGENIC MICE FOR THE DETECTION OF GENE-MUTATIONS IN GERM-CELLS

Recently-developed transgenic models have provided unprecedented acces s to rodent somatic and germ line tissues for the study of gene mutati on in vivo. While mutations in germ cells are considered an important aspect of any regulatory assessment of the risks posed by chemicals, c urrently-available conventional tests, which involve the study of thou sands of offspring make it impractical to test large numbers of chemic als, for the induction of inherited gene mutations. When effects in ge rm cells per se, rather than offspring are acceptable targets, transge nic mouse assays may provide a practical alternative. As part of an in ternational collaborative study to begin to determine the reliability, efficacy, and role of such assays, lacZ transgenic mice (Muta(TM) Mou se) were treated with single i.p. doses of ethylnitrosourea (ENU), met hyl methanesulfonate (MMS), and isopropyl methanesulfonate (iPMS), and mutant frequencies determined using phenyl-beta-D-galactoside (p-gal) positive selection. For studies using germ cells, the selection of sa mpling times and target cells is crucial. Spermatagonial stem cells an d cells in post-spermatagonial stem cell stages are the critical targe t cell populations of regulatory importance. Cell populations within t hese categories were studied by sampling germ cells isolated from semi niferous tubules and spermatozoa from the epididymis at 91 days and 25 days after treatment. The data show that ENU and iPMS induced mutatio ns in post-spermatagonial stem cells and spermatagonial stem cells. Ho wever, MMS did not induce mutations in either cell type, or at either sampling time, at doses approaching lethality. This result is possibly because MMS induces preferentially large lesions and chromosomal aber rations (as opposed to point mutations), which are not readily detecta ble with bacteriophage-based shuttle vectors. Since MMS-induced specif ic locus and dominant lethal mutations are induced only after the mid- spermatid stage, it is also possible that the timing used missed this effect. While the ENU and iPMS data in this study demonstrate the suit ability of the lacZ male transgenic mice for the study of gene mutatio ns in post-spermatagonial stem cells and spermatagonial stem cells by sampling cells isolated from seminiferous tubules at selected times af ter treatment, the MMS results do not answer fully whether transgenic mouse mutation assays can detect mutations resulting from lesions indu ced after the mid-spermatid stage when most cellular processing is ret arded. Nevertheless, it appears clear from presently available informa tion, that the bacteriophage-based lacZ transgenic model is suitable f or the detection of gene mutations in spermatogonial stem cells, sperm atocytes, and early spermatids.