| Melamine and related compounds(MARCs)have been a focus of food safety since the "poisoned milk powder incident" in 2008.Studies on the chemical properties,metabolism,biological effects,and toxicology of MARCs have shown that despite the acute toxicity of Melamine(MEL)and cyanuric acid(CA)are lewow,these compounds are difficult to be metabolized by mammals.MEL and CA are usually discharged through urine,but when they are used in high doses,these compounds will have significant renal stoxicity and also have effects on the heart and other organs.Therefore,it is particularly necessary to conduct in-depth studies on the biological and toxicological effects of MEL and CA,such as how does it work in vivo and biomolecules,and whether synergies will occur if MARCs coexist.We need to answer the questions from physiological,biochemical,pathological and other aspects.The primary biological target for the exposure of MARCs in the environment is micro-organisms.The purpose of this study is to understand the apparent biological effects of MARCs alone and mixed exposures on the environmental microorganism--Saccharomyces cerevisiae’s apparent effect and mechanism of action.Using flow cytometry and gas chromatography to detect the growth and physiological changes of Saccharomyces cerevisiae,the composition and content of phospholipid fatty acids in cell membranes which under the stress of MARCs.The expression of eGFP gene in Saccharomyces cerevisiae was detected by real-time fluorescence quantitative PCR.The results showed that the membrane permeability of Saccharomyces cerevisiae increased under the stress of MARCs and the proportion of stained cells increased after cultured for 24 h,and there was a positive correlation with the change of MARCs concentration.For 0.25 mg/mL mixing in the exposed system,when MEL and CA were present at the same time and the volume ratio reached 1:1,the change of cell membrane permeability of Saccharomyces cerevisiae reached the maximum,which was significantly higher than that of 0.25 mg/mL single component exposure system;7 main fatty acids were detected by gas chromatography.The main fatty acids of the cell membrane of Saccharomyces cerevisiae were unsaturated fatty acids C16:1.With the increase of MEL and CA concentrations,the content of unsaturated fatty acids increased significantly,and the content of saturated fatty acids decreased significantly.The ratio of relative content of unsaturated fatty acids and saturated fatty acids increased from 1.4-fold to 3.4-fold as compared with the control group,and gradually increased with increasing concentrations of MEL and CA;Detection of Saccharomyces cerevisiae eGFP by flow cytometry the expression of protein was vgnegatively correlated with the change of MARCs concentration.The higher the MARCs concentration,the higher the inhibition rate of eGFP protein expression.The inhibitory rate of eGFP protein expression when the concentration of MEL and CA was 2.0 mg/mL alone was 17.07.%and 12.92%,0.25 mg/mL MEL and CA alone,the inhibition rate of eGFP protein expression were 9.70%and 8.35%,respectively,and when 0.25 mg/mL MEL and CA mixed exposure conditions,When the ratio reached 1:1,the inhibition rate of eGFP protein expression reached 20.45%.The real-time quantitative PCR technique was used to analyze the relative expression level of eGFP gene under MARCs stress,which was consistent with the expression level of eGFP protein.The above studies showed that the membrane of Saccharomyces cerevisiae was firstly impacted under MARCs stress,resulting in changes in cell membrane permeability and fluidity,and caused a certain degree of oxidative damage to Saccharomyces cerevisiae eventually leading to apoptosis,MEL enters the intracellular interference of Saccharomyces cerevisiae.The normal metabolic process also has a certain degree of inhibition on its protein expression and related gene expression.The assessment of toxicity of MARCs have certain reference value in both the cytotoxicity and genotoxicity. |
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