The main goal of this proposal is to test the hypothesis that metallothionein (MT) is able to effect the level of the mutagenic events induced by genotoxic carcinogens. There is some contradictory data concerning the sensitivity of cells with varying levels of MT expression sensitivity to the toxic effects of carcinogens and chemotherapeutic agents. However, no studies have yet been carried out on mutation frequencies in such cells. We have developed a series of Chinese hamster V79-transgenic cell lines containing single copies of the E. coli gpt gene. One of these, G12, has been shown have a target gene (gpt) which is normally mutable by alkylating agents and highly mutable by agents causing oxidative damage (e.g. x-rays) which do not produce many gene mutations in most systems. Like many permanent cell lines, G12 has a low level of constitutive and induced MT expression. We have used G12 cells to create MT-overproducing cells by transfection of a mouse MT-I gene in a pBPV vector. Preliminary results show that the MT transfectant with the highest level of expression has an elevated spontaneous mutation frequency compared to its parent, G12. However, these results are confounded by the presence of extrachromosomally replicating plasmid, which might confer genetic instability. We propose to construct transgenic lines, using G12 as host, which contain stably integrated copies of mouser MT-I. In addition, we will create an MT anti-sense expression vector in order to knock out host MT-I expression. These lines will be used to measure the spontaneous mutation frequency as well as the induced mutagenesis after treatment with a number of metal (crystalline nickel sulfate and calcium chromate), physical (UV and x- rays) and organic (MNU and MNNG) carcinogens and chemotherapeutic agents (cisplatin, bleomycin, mAMSA). The abilities of arsenite, Cd(II), and Pb(II) to act as comutagens will also be evaluated. If effects on mutagenesis or comutagenesis are found to vary with MT expression, effects of MT levels on DNA repair enzymes (which require zinc) will be studied in permeabilized cells.
