This laboratory has interest both in basic molecular mechanisms of mutation and in using rapid and inexpensive microbial bioassays to identify and quantitate the genotoxic action of environmentally significant mutagen/carcinogens. Most of these assays suffer from a major flaw-the microbes being used to achieve a mutagenesis endpoint do not metabolize xenobiotics in the sam fashion that mammalian cells do. Identification of activation requiring genotoxins and extrapolation of microbial data for use in human impact assessment are therefore impossible. The metabolizing enzymes in mammalian liver cells can be extracted (S9) and added with a cofactor regeneration system and buffers to assay mixtures. Enzymes levels can be increased and composition can be changed by inducing the tissue- donating animals with various chemicals before extracting the microsomes. Conditions of assay such as protein concentration and temperature must be optimized to obtain peak mutagenesis for each chemical. These enzymes are also temperature sensitive and short lived in activity, making manipulation tedious and technically demanding. Those labs doing significant amounts of testing and using a high level of automation, such as ours, find most of the testing with enzyme being done to a large extent by hand. To circumvent this problem cloned P450 enzyme cDNAs, in an appropriate expression vector, could be used to transform our test strains. Then the same strain could be used to test with and without enzyme, inducing synthesis of the enzyme in the latter case. Rat and bacterial cDNA for P450 enzyme forms has recently been cloned and expressed at least to some extent in E. coli and an oligonucleotide probing sequence is known for at least one enzyme subgroup. This subgroup is the one specifically induced by alcohol, one of our research areas of interest. This proposal is therefore to place a plasmid-borne, P450 metabolizing system in a series of repair deficient, mutagenesis sensitive strains of E. coli, to begin testing of these new constructs for feasibility os use in our semiautomated testing system and to investigate the specificity and interactions of the enzyme in alcohol containing mixtures. Students can be involved in every aspect of research and publication.
NIH ExPorter
NIH ExPorter