Assessment Of Genetic Toxicity Interactions Of Diverse Ternary Mixtures Using Response Surface Analysis Method Based On The Dual Fluorescent Yeast Cells Regulated By RAD54 Promoter

The traditional method of bioassay for chemical mutagens has been limited to single chemcial.How to bioassay the mutagenic effect of two or more chemical compounds,that is,whether it will produce additive,synergistic,and antagonistic effect,until now,there are few studies reported subject.However,human are exposed to a variety of chemical mutagens pollutants exist in the environment,the mechanism of interaction among the chemcials in vivo are complex,producing antagonistic,additive,unrelated or synergistic effects.Response surface analysis is a new method of interaction at compounds research,including experimental design,modeling,inspection model suitability,seek the best combination of the condition of test method and technology;Through the process of regression fitting and the response surface and contour,can easily find out corresponding to each factor level response values.1.The selection of RAD54 promoterTwo yeast enhanced green fluorescence protein(yEGFP)yeast reporter vectors,pR1558-yEGFP and pR406-yEGFP,which are regulated by two RAD54 promoters containing 406-bp and 1558-bp DNA sequences,respectively,were constructed using molecular biological techniques and transformed into yeast for the screening of genotoxins.The constructed biosensors were named W303-1A/R1558-yEGFP and W303-1A/R406-yEGFP.To quantify biosensor performance,the both transformed yeast cells were exposed to multiple doses of genotoxins including methylmethane sulfonate(MMS;a DNA alkylating agent),4-nitroquinoline-N-oxide(4-NQO;a DNA cleavage agent),5-fluorouracil(5-Fu;an inhibitor of polymerases and topoisomerases).The yeast bioassay performance was analyzed using fluorescence-activated cell sorting(FACS)and Multi-Mode Reader in a 96-well black microplate.The results showed that the cytosensors have significant dose effect relationship.However the observed W303-1A/R1558-yEGFP dose-effect relationship was more obvious and the maximum inductions were 5.96-fold(MMS),2.19-fold(4-NQO)and 2.71-fold(5-Fu);the corresponding values for W303-1 A/R406-yEGFP were 2.53-,1.79-and 1.91-fold,respectively.At the same time,biassay results from FACS are more sensitive than that from Multi-Mode Reader.Therefore the entire RAD54 promoter was selected to construct recombinant yeast cells for genetoxic study.2.Construction of dual fluorencent recombinant yeast cells regulated by whole sequence of the RAD54 promoterSince the luminescence of cells is not only related to the induction of mutagenesis,but also affected by the number of cells and the state of the cells,the DsRed-Express2(red fluorescent protein)was used as the second signal to construct dual signal yeast whole-cell biosensors with red and green fluorescent proteins,which were regulated by R1558 promoter.When the recombinant yeast cells are treated by the chemical mutagenesis,the yEGFP/DsRed-Express2(relative luminosity)had a significant dose effect relationships with the chemical mutagens.3.The interaction mutagenic study based on the response surface analysis(1)Initially,the chemical mutagens were evaluated individually by the recombinant dual signal flurencent yeast cells for the single factor experiment study in order to establish individual chemical concentration-response data.The flurencent intensity of yEGFP and DsRed-Express2 was measured by a multifunctional luminescence analyzer,and then calculate the relative flurencent density.The concentration of each chemical mutagenesis threshold was determined according to the dose-effect relationship.(2)The three interaction parameters(?123,P = 0.011),statistically significant,from MMS,Chlorambu and Cis-Platinum were found indicating that there is interaction among the chemicals.Further analysis showed that the parameter(?12,P = 0.113)from MMS and Chlorambu,that(?23,P = 0.613)from Chlorambu and Cis-Platinum were not statistically significant,while theinteraction((?13,P<0.005)from MMS and Cis-Platinum was significantly obvious.Then,by analyzing the contours of the response surface,it can be seen that there is no linear relationship between MMS and Cis-Platinum.With the increase of the concentration curve,the curve is bowed outward,and then the bow is oriented toward the origin.Therefore in the low concentration MMS and Cis-Platinum exhibited antagonism,but in high concentrations synergies.(3)The combination genotoxicity study of 5-fluorouracil,camptothecin and hydroxyureafound that the interaction parameter(?123,P<0.001)was statistically significant,indicating that there is interaction among them.The interaction(?12,P = 0.008)from 5-fluorouracil and hydroxyurea,the parameter(?13,P = 0.006)from camptothecin and hydroxyurea were found to have the different degrees of interaction by further analysis.From the curve of the response surface curve,it can be seen that there is no linear relationship between camptothecin and 5-Fu.With the increase of the concentration curve,the curve shows the tendency of the arch to the origin,showing that there is synergistic effect between the two chemicals.