Integration of leave-one-out method and real-time live cell reporter array system to assess the toxicity of mixtures

Abstract

The ever-increasing number of chemicals and complex mixtures demands a time-saving and cost-effective platform for environmental risk assessment. However, there is limit promising tool for evaluating the contribution of each component to the total toxicity effects of the mixture. Here, four widely distributed environmental pollutants with different mode-of-actions, i.e., cadmium chloride (Cd), nitrofurazone (NFZ), triclosan (TCS), and tris(2-chloroethyl) phosphate (TCEP), were selected as components of artificial mixture. Integration of leave-one-out method and high-dimensional live cell array system was used to explore relative contribution of each component from the mixture. A quaternary mixture (All_4_chems) and four ternary mixtures (Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP) were investigated by Escherichia coli (E. coli) live cell array system with 90 environmental stress genes modified by green fluorescent protein (GFP) expressing reporter vectors. E. coli cytotoxicity tests demonstrated that TCS has antagonism effect with other three chemicals (Cd, NFZ and TCEP), while it was additive effect in other three binary combinations. A total of 26, 23, 13, 31 and 23 genes were significantly altered with fold-change greater than 2 over the 4 h exposure by All_4_chems, Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP, respectively. Clustering analysis based on time-series gene expression patterns and transcriptional effect level index (TELI) showed that Leave_TCEP has similar profiles with All_4_chems, demonstrating TCEP has the least contribution among four components to the quaternary mixture. Leave_NFZ has the least number of significantly altered genes, implying NFZ has the largest toxicity effect contribution to the quaternary mixture. The relative contribution in different pathways indicated that Cd has the most contribution to the mixture in redox stress, while TCS has the least contribution in DNA stress pathway. Collectively, our results demonstrated the utility of high-dimensional toxicogenomics data and leave-one-out method in prioritizing the relative contribution of each component in mixture.