Organs coming most closely into contact with ingested ethanol (buccal cavity, pharynx, larynx, esophagus) are primarily subject to increased cancer risk. The studies proposed within this application are oriented toward understanding the mechanism for the observed mutagenic effect of ethanol in the RK mutatest. This test is a relatively new, short-term assay system for detecting mutagenic activity. The test system is based upon the observation that a segment of the bacteriophage lambda genome responsible for phage replication, and integrated within the E. coli chromosome, places the viability of the cell under the control of lambda replication initiation. Induction of replication from the lambda fragment generates multiple replication forks and produces cell death (replicative killing, RK+ phenotype). Mutation of the lambda replicator region permits escape from replicative killing. Although the RK system cannot compete with rapid mutational systems for determining mutagenic spectrum, the RK mutatest may have an advantage to simpler systems in being able to identify agents which induce large chromosomal deletions, gene rearrangements, or for studying gene amplification. We have determined that transient cell exposure to greater than 18% ethanol is mutagenic in one of the RK+ selector strains [SA500(lambda cI857 delta 431] that includes on the integrated lambda fragment the lambda genes int - kil (encoding several genes involved with general and site-specific recombination). Ethanol unambiguously enhanced the proportion of RK- survivors with large deletions of the lambda replicator fragment. Ethanol was not mutagenic to three isogenic stains deleted for genes int - kil. The experiment proposed in this application will determine which of the lambda genes in the int - kil interval are necessary for demonstrating ethanol mutagenicity. Four new RK+ selector strains are to be constructed that are deleted for potions of the int to kil region. These RK+ strain constructs will be used to assess ethanol mutagenicity. RK mutatest protocols developed previously are to be employed. We believe this experiment can pinpoint the genes encoding metabolic functions that are capable of generating large deletions (when perturbed by exposure of RK+ cells to ethanol).
