Development, Optimization And Preliminary Application Of Targeted Analytical Toxicology In Environmental Toxicology

Toxicology, especially environmental toxicology, focuses on the occurence of chemicals in organisms and their subsequent effects on organisms. One of the most important aims of most toxicological studies is to establish the links between chemical exposure and the biological/molecular responses in organisms. In order to achieve this goal, concentrations of chemicals and the responses (often referred as biomarkers) should be both determined. In modern studies, chemical/s and biomarker/s are usually separately determined, which consumes much time, cost and labor. In addition, the limitation of certain bioassays for biomarkers also limits their application, for example, the corresponding antibodies are necessary for the determination of protein biomarkers in immune-based bioassays.The development of targeted proteomics allows the simultaneous determination of levels of the chemicals and protein biomarkers in the same specimen sample. Targeted proteomics is a new method which has been developed recently to quantify a subset of proteins of interest in a sample. Selected proteins are first enzymatically digested into peptides which are then quantified by LC-MS/MS, with high sensitivity, quantitative accuracy and reproducibility. In targeted proteomics, signature peptides digested from selected proteins are recommended to be quantified by LC-MS/MS, especially triple-quadrupole instrument. Many environmantal pollutants are also ideal to be quantitatively analyzed by liquid chromatography tandem mass spectrum. Thus, in theory, it is feasible to simultaneously measure these two types of compounds by LC-MS/MS. However, such attempts have not yet been reported in the literature.We reported a method, named as Targeted Analytical Toxicology (TAT), for the simultaneous determination of levels of chemicals and protein biomarkers in organisms by UPLC/ESI-MS/MS on the basis of targeted proteomics. The TAT method was successfully applied to detect the concentrations of 17a-ethynylestradiol (EE2) and EE2-induced vitellogenin (a biomarker for estrogens) in male zebrafish exposed to EE2. A significant positive relationship was observed between the EE2 concentrations and VTG levels (p<0.05). The dose-response curve based on the concentrations of chemicals in organisms instead of the nominal concentrations was more reasonable in toxicological studies.We also optimized TAT method after development of the method. Signature peptides with different lengths and trypsin digestion sites were sythesized in order to trace the trypsin digestion efficiency. And the experimental results showed little difference among the signature peptides with different lengths and trypsin digestion sites. We also optimized the pretreatment method to increase the recoveries of EE2. The results indicated that SPE pretreatment method increased the recoveries of EE2 compared to the 3kDa spin unit pretreatment method, and therefore improved the method performance.After development and optimization, TAT method was then applied into a case of environmental toxicology. We characterized the effects of organophosphate flame retardants (OPFRs) on three protein biomarkers (interleukin 6 (IL-6), tumor necrosis facor alpha (TNF-a) and oncoprotein p21 ras) in human pulmonic A549 cells. The method was employed to simultaneously quantify OPFR/s and the three proteins. Although TAT can analyze several OPFRs and several protein biomarkers simultaneously, the overexpressions of IL-6, TNF-alpha and p21 ras were not detected in A549 cells exposed to OPFR/s. Meanwhile, ELISA kit was also used to validate the results by TAT. Both results suggested that OPFR/s might not induce the overexpressions of these proteins in A549 cells exposed to OPFR/s.This TAT method allows us to quantify concentrations of chemicals and proteins in a sample at the same time by the employment of the same sample preparation method and analytical instrument. It is expected for TAT to have broad application in toxicology, especially in environmental toxicology, due to its high sensitivity, accuracy, reproducibility, high throughput, and the savings of time, cost and labor.