It is important to detect genotoxic chemicals for environment protection and cancer prevention. A genotoxic testing system of RNR3-lacZ based on yeast RNR3 transcriptional response to DNA damage has shown some advantages: this testing system has not only traits of eukaryotes, but also traits of prokaryotic bacteria for easy culture and gene operation, which can detect various kinds of genotoxic chemicals and appears to be more sensitive than other similar tests in microorganisms, such as bacterial SOS chromotest assay, and is complementary to the Ames Test. Due to the permeability of yeast cells and its simpler metabolic activation system, this system has been compromised for practical applications. In order to apply this testing system for environmental detection, further enhancement of detection sensitivity and spectrum is desired. In this desertation, we have attempted genetic manipulation in the following areas:
1. Enhance permeability of yeast cells:
Cell wall, cell membrane and the PDR pathway (pleiotropic drug resistance) limit the accumulation of genotoxic chemicals in the cell. We try to alter the cell wall and membrane structure, or inhibit the membrane efflux function through gene deletion to enhance the RNR3-lacZ system sensitivity. The results show that inactivation of both CWP1 and CWP2 gene encoding cell wall mannoproteins has profound effects on the cell wall structure and permeability, and markedly enhances the system sensitivity to high molecular weight compounds. Menawhile, inactivation of both SNQ2 and PDR5 genes encoding membrane efflux proteins enhances the system sensitivity to compounds with any molecular weights. We hope to incorporate two pathways to further enhance the systeme sensitivity.
2. Enhance metabolic activity of yeast cells:
Many chemical carcinogens require metabolic activation before they show genotoxicity. Because of lack of the key metabolic enzyme cytochrome P450 (CYP), the yeast is unable to activate these chemicals. We genetically engineered the yeast to produce human CYP1A1 or 1A2. The gained metabolic competent yeast can activate aromatic amine 2-aminofluorene (2-AF) or 2-acetyl aminofluorene (2-AAF) to induce RNR3-lacZ, but failed to activate benzo[a]pyrene (BaP). However, BaP can be metabolically activated by yeast expressiong human CYPlAl as whole cell biocatalyst and show mutagenesis in Ames test. In order to achieve a log-term activation, we created a RNR3-EGFP system in a hope to accumulate sufficient amount of CYP4501A1 for metabolic activation of BaP that leads to induction of RNR3.
3. Enhance oxidative stress of yeast cells:
Many chemicals damage DNA through the release of reactive oxygen species (ROS), which maybe cause cancer or aging. However, the yeast RNR3-lacZ show poor sensitivity to DNA oxidative agents. Inactivation of YAP1, a key transcriptional activator in the oxidative stress response, markedly enhanced RNR3-lacZ sensitivity to oxidative agents. Furthermore, inactivation of YAP1 or SKN7, another transcriptional activator in the oxidative stress response, also enhanced RNR3-lacZ sensitivity to MMS. MMS as been regarded as an alkylating agent and not previously reported to cause DNA damage through ROS.
In summary, we had enhanced sensitivity of the yeast RNR3-lacZ genotoxic testing system through altering different biological pathways and observed some new phenomena, which provide more research groundwork for this as well as other similar testing systems into practical applications. This study also provides insights into the mechanisms of cellular response to DNA damage in eukaryotes