Creation and improvement of a yeast RNR3-lacZ genotoxicity testing system
Date
2003-01
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ORCID
Type
Degree Level
Masters
Abstract
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.
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Degree
Master of Science (M.Sc.)
Department
Toxicology
Program
Toxicology