| DNA adduct is one of the most important biomarkers. It is the product of DNA covalently binding with the electrophilic group of compound, and is the most important and commonly mode of DNA damage. Detection of DNA adduct can not only provide the exact interior dosage but also help to illuminate the toxicology mechanism. The research brings DNA adduct back to the centre of research interest. Sulfur mustard (SM) is well known to be a highly toxic and mutagenic warfare agent, featured with synthesis facile, hypertoxicity, security and therapy difficult.Sulfur mustard possesses two electrophilic carbon atoms and its chemistry and metabolism are dominated by their reactions with macromolecules (protein, DNA, RNA et.al.) via the episulfonium ion. The mechanism of its action with regard to vesicancy is unknown but is assumed to result from its reactions with macromolecules. Despite the lowest content and shorter half-time, DNA adducts show important biological significance.SM reacts with DNA to produce four kinds of mono- and bifunctional adducts. These adducts are formed preferentially at the N7 position of guanine (60-70%), so N7-HETEG is considered to be suitable for the retrospective detection of SM. Even though the other three adducts (O6-HETEG, Bis-G and N3-HETEA) are comparatively rare, they have great relationship with biological endpoints, such as misreplication, stall replication and finally induce double strand breaks. So the research on the DNA adducts may help to clarify the toxicological mechanism and lay a foundation for the research of SM prevetion and therapy.For SM retrospective detection and the toxic mechanism illumination, two sensitivity detection methods were developed using HPLC-MS/MS in MRM mode. The methods were used to the detection of the biological samples in vitro and in vivo. The metabolizing behavior and distribution of adducts were evaluated in SD rats administrated with SM percutaneous and ip.This dissertation consists of four chapters. The first chapter is the introduction. In this chapter, we summarized the biological significant and detection methods of DNA adducts. The research progress of the SM-DNA adducts was focused on. The detection methods and the important role in retrospective detection and toxic mechanism illumination were sumarized. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.The second chapter was about the synthesis and identification of standards. In this chapter, nine pure standards were obtained, which include four DNA adducts, four deuterium labeled internal standards (IS) and N7-benzylguanine. The structure identification and the purity check were performed with 1H NMR and LC-MS.The reactive sites of guanine and adenine are N7 and N3 respectively, N7-HETEG and N3-HETEA were direct synthesised from less toxic material- half mustard in organic homogeneous system. Reactivity of O6 is remarkably less than that of guanine N7, so O6-HETEG can not get from half mustard simply covalent binding to guanine or guanosine. 6-cholorguanine and nontoxic compound- thiodiglycol were used to synthesis O6-HETEG skillfully. Bifunctional SM adduct was gained according to the procedure of reference. Before purified with preparation chromatography, crude product should be washed with diluted acid repeatedly to get rid of the main byproduct-N7 monofunctional adduct.In order to pave the way for synthesis of deuterium labeled internal standards, synthesis method of deuterium labeled sulfur mustard and thiodiglycol were established.All of the puritis of the nine standards were over 95%, and the deuterate percentage of internal standards were over 99%.The third chapter is about the development and verification of analysis methods of SM-DNA adducts.Two detection methods were developed. The first one aimed at detection of the highest aboundant lesion N7-HETEG, using high performance liquid chromatography– positive electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS), working in multiple reaction monitor (MRM) mode and N7-benzylguanine act as IS. The limit of detection was hundreds pg/mL in various biomatrices, viz. one adducts per 107 nucleotides. Validation results showed that the method has satisfactory sensitivity, precision, and accuracy. N7-HETEG in human whole blood treated with 2μmol/L SM in vitro was detected successfully. There is dose-dependent relationship between N7-HETEG and SM dosages, so N7-HETEG can act as the biomarker of SM exposed.Simultaneity determination method was developed at first time using ultra high pressure liquid chromatography-positive electrospray ionization tandem mass spectrometry (UPLC-ESI+-MS/MS), quadrupole and ion trap tandem working in MRM mode, which provides higher sensitivity. Higher selectivity and specificity were achieved by isotope dilution method. The limits of detection of N7-HETEG, O6-HETEG, Bis-G and N3-HETEA were 1 pg/mL, 0.2 pg/mL, 5 pg/mL and 9 pg/mL respectively, nearly one adduct per 109-11 nucleotides. But it need higher synthesis technic and more expensive instrument。DNA adducts in SD rat derma in vitro were detected with this method, result revealed the linearity dependence between DNA adducts content and exposed dosage.The last chapter is about the development of animal models and the metoblizing behavior of SM-DNA adducts. Adult male SD rat was used as the experiment animal, treated with different SM dosages by percutaneous and intraperitoneal injection (ip) exposure. At the given time postdosing, the rats were euthanized with urethane by intraperitoneal injection, the blood was collected. Livers, lungs, spleens and kidneys were immediately isolated and weighed. A portion of these tissues were prepared for histopathological evaluation, the rest were used to DNA adducts detection.The relative spleen weights of SD rats dermal exposed to 45.0 mg/kg SM decreased sharply and had less volume of blood, because higher dosages exposure led extensive damage of haematogenous system. The histological changes of the lung treated by both of two routes were remarkable.The results of detection revealed that the DNA adducts in derma, tissues and blood were all detectable even at 14 or 21 day postdosing, the dose and time dependence were remarkable, which showed DNA adducts can act as the biomarkers for retrospective detection. The blood maybe perform the main metabolizing medium transmit SM to tissues. The decreased order of content in derma was N7-HETEG, Bis-G, N3-HETEA and O6-HETEG, but the content of O6-HETEG was larger than that of N3-HETEA in blood and tissues. The fact that higher contents of O6-HETEG lied in lung and spleen showed that O6-HETEG has great relationship with SM damage. According to the different percentage of DNA adducts in blood and tissues, the conculsion was achieved: N7-HETEG, N3-HETEA and Bis-G could be the excellent exposed biomarker instead of the effect biomarker. O6-HETEG maybe is the important effect biomarker of SM damage.In conclusion, two sensitively and selectively methods were developed for analysis the SM-DNA adducts. The trace SM-DNA adducts in biosamples can be detected successfully. The metabolizing behavior research and retrospective detection were layed foundation. Detecting four SM-DNA adducts simultaneity reflected the distribution and metabolizing process truthly. The relationships between adducts and exposure dosage, damage effect were opened out in molecular level. New thoughtway was provided for the research of SM toxicologically mechanism.
|
PDF Full Text Request