Creation and improvement of a yeast RNR3-lacZ genotoxicity testing system
A variety of environmental toxicants can damage DNA and thereby produce congenital malformation and cancer. In order to evaluate the genotoxic effects of environmental agents, numerous genotoxicity testing systems have been developed. These tests either directly assess the genetic alterations (e.g., the Ames test) or indirectly measure the cellular response to DNA damage (e.g., SOS Chromotest). With the knowledge obtained from studying molecular mechanisms of DNA damage and signal transduction in Saccharomyces cerevisiae, a sensitive and stable genotoxicity testing system was developed based on the induction of a S. cerevisiae RNR3-lacZ reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. The tested agents include known carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals to ionizing radiation as well as some known non-genotoxic agents. All the tested known genotoxic agents were able to induce RNR3-lacZ expression at a sub-lethal dose. In particular, a potent colon carcinogen, 1, 2-dimethyl hydrazine, was not detected as a mutagen by a standard Ames test, but was able to induce RNR3-lacZ expression. In contrast. both non-mutagenic and non-genotoxic chemicals tested were unable to induce RNR3-lacZ expression. The sensitivity and inducibility of three well-characterized yeast DNA damage-inducible genes have been compared and it was found that RNR3 is more sensitive than RNR2 and MAG1. The effects of agent dose, post-treatment incubation time and cell growth stage on RNR3-lacZ expression were also determined and optimized. In order to create a stable and user-friendly testing system, the RNR3-lacZ cassette was integrated into the yeast genome to demonstrate that its inducibility is indistinguishable from that of the plasmid-based studies. Although the sensitivity of RNR3-lacZ testing is comparable to that of the Ames test and SOS Chromotest, it was reasoned that the sensitivity might be further improved by using yeast strains defective in certain DNA repair pathways. Hence, several deletion mutant strains from different repair pathways were used as host strains for the RNR3-lacZ test. It was found that the mag1 null mutation specifically enhanced the sensitivity of RNR3-lacZ test in response to DNA alkylating agents such as methyl methanesulfonate and ethyl methanesulfonate, while the rad2 null mutation enhanced the sensitivity of this system in response to ultraviolet (UV) radiation and a UV mimetic agent 4-nitroquinoline 1-oxide. In the rad2 null mutant, RNR3-lacZ induction was also more sensitive to MMS than that in wild type strain at low dosed. In summary, it appears that the enhancement of RNR3-lacZ induction is agent and repair specific, although inactivation of the nucleotide excision repair pathway may affect a broad range of testing agents which can be incorporated into the RNR3-lacZ test.
Master of Science (M.Sc.)