Study On The Biological Effect Of The Newly Discarded Animal In

Sulfur mustard(SM) is one kind of vesicant with powerful alkylating effect, and could rapidly penetrate skin, ocular and lung bronchus mucous membranes and react with numerous nucleophiles in vivo. After World War II, a large amount of chamical weapons abandoned by Japanese army, which were major consisted by SM, are continuously threaten the life and property of people in China. In recent years, as the international extreme nationalism and religious resurgence, the possibility of SM used to terrorist attacks and asymmetric warfare is increasing. So researches on SM damage mechanism and control methods are still significant and valuable.Since the first sythesis, the study of SM has lasted over one century. There were many hypothesis were proposed to investigate the lesion of SM exposure, but the certain mechanism is unknown, yet. Because of the lipophicity, SM could penetrate the surface barrier of body soon after exposure, invade into tissues and circulatory system, and alkylate with most of life molecules, such as DNAs and proteins.These alkylation can affect DNA synthesis and repair, interfere with the enzyme activity and cell metabolism, eventually leading to cell apoptosis, inflammation, immune response and metabolic disorders. The symptoms of SM damage has incubation period and can be observed hours after exposure. Because of the complexity and diversity of symptoms, there are no special treatments towards SM damage. Therefore, SM is still a chemical warfare agent with difficulty to prevent and cure.Our lab previously established a LC-MS method to detect the SM prototype of after derivatization. And the distribution of the SM prototype in the tissue of rats was studied. The results suggested that there was high level of SM prototype existed in adipose tissue(AT). Consequently, the phenomenon of SM accumulation in adipose was observed and confirmed. By the establishment of a SM-DNA adduct quantitative detection method, the distribution of SM-DNA adduct in tissue of rats was also researched. The data showed that the levels of SM-DNA adduct were relatively high in lipid-rich tissues, such as bone marrow, and brain. But the contents of SM-DNA adduct were relatively low. It prompt that lipotropy environment may have a significant influence on the formation adduct.Based on the early research of our laboratory, this paper focus on the toxicity effect of the accumulation of SM in adipose by evaluation the formation of SM-DNA adducts, cell survival and lesion in exposed adipocytes in vitro, as well as evaluation of the fat-body ratio, pathological injury of AT from exposed rat in vivo. The preliminary biological significance of as well as the m RNA and protein level of adipokines were also evaluated in rat SM-exposure model. The toxicity effect and biological significance of the accumulation of SM in adipose were also discussed by evaluation of the adipokines change both in m RNA and protein level. Full paper is divided into five chapters.The first chapter is introduction. In this part, the physical and chemical properties, sythesis and implyment history, and the damage mechanism research progress of SM were introduced. The novel discovery in the function of AT and adipokines were described. Eventually, the purpose, brief content and innovation points of this paper were exhibited.The second chapter is the establishment of SM-expoure model in vitro. Utilizing the original preadipocytes separated from rat AT, the differentiation process for mature adipocytes was optimized. Following the optimized process, human mature adipocytes(HA-s) were differentiated from cell line of human subcultaneous preadipocyte(HPA-s) with a high differetiation ratio above 80 %. As experimental control, three kinds of cell lines derived from major injury tissue of SM exposure were cultured, including human keratinocyte Ha Ca T, human hepatocyte L-02 and human lung fibroblasts HLF. And the SM exposure IC50 values were detected by CCK-8 kit. The results showed that the IC50 value of HA-s(484.3±22.7 μM) was obviously higher than the other four kind of cells(77.5±5.5 μM for Ha Ca T, 69.9±4.3 μM for HLF, 73.8±3.7 μM for L-02 and 62.5±0.4 μM for HPA-s), which suggested that mature adipose has higher survival post SM-exposure.The third chapter is detection of SM-DNA adducts from SM exposed cells in vitro. Based on the established UPLC-MS/MS method for SM-DNA adducts simutaneously quantification, validation was performed. Results showed that the current method has good selectivity, linear range, accuracy and precision. The limits of quantification were 0.02, 0.05 and 0.1 ng/m L for N7-HETEG, Bis-G and N3-HETEA, respectively. The matrix effection and recovery were all conform the requirement. It indicated that the current method could be used for dectection of samples from SM exposed cells in vitro. The formation of SM-DNA adducts in five cells were quantified, and the dose-dependent and time-dependent trend were analyzed. As shown in data, the formation of SM-DNA mono-adduct was faster than bi-adduct, and the concentration order of adducts were N7-HETEG > Bis-G > N3-HETEA. By conparision, the differences in adduct levels among cell lines exposed in vitro were less pronounced than in vivo. The percentage of Bis-G in HA-s were more than other cells. The time to peak and half-life time of adducts in exposed HA-s were also the longest. These results indicated that the mature adipocytes have resistance against SM exposure.The fourth chapter is the influence of SM to cell function post exposure in vitro. Here we used fluorescent dyes of Hoechst 33342, Cell Mask, Mito Tracker and Lyso Tracker, and antibodies of Mn SOD and p H2 A.X to stain cells. Through high content screening method, the exposure dose-dependent and time-dependent trend of cell damage were observed in exposed HPA-s and HA-s, including cell morphology damage from the nucleus and cytoplasm, mitochondrion and lysosome damage injury, oxidative stress and DNA damage. Results showed that cell lesions in exposed HA-s were lighter than in exposed HPA-s, including oxidative stress and damage of mitochondrion, lysosome and DNA. It certified the resistance of muture adipocytes against SM exposure. And then, the culture supernatant from exposed HA-s and HLF were collected to cocultured with exposed cell lines of Ha Ca T, HLF and L-02. By CCK-8 method, the influence of culture supernatant to exposed cells were evaluated. Data showed that co-cultured with supernatant of exposed HLF, IC50 values of exposed cells would more than co-cultured with medium. But the values of IC50 of exposed cells co-cultured with supernatant of exposed HA-s, would less than co-cultured with medium. By the increase of SM exposure dasage in HA-s, the IC50 values of co-cultured cells post exposure were decrease. This implied that adipokines and secondary toxicity of accumulated SM in adipose tissue may inhibit the growth and activity of other kinds of cells. And it also reminded the importance to eliminat the accumulated SM in adipose tissues to decrease the secondary lesions.The fifth chapter is the influence of SM exposure to the function of ATs from rats. The time-dependent trend of rat weight and fat-body ratio were evaluated in SM exposed rats. Results showed that ATs were rapidly consumed, which proposed the posibility that the accumulated SM in AT would release due to tissue consumption.The lesion degrees of subcutaneous AT, perirenal AT, epididymal AT and brown AT caused by SM exposure were assessed by H&E stained pathological section. From photographs, the lesions of adipocytes in white AT, including subcutaneous AT, perirenal AT and epididymal AT, were not significant. But SM exposure caused serious damage in brown AT, where the brown adipocytes were tend to transform into white adipocytes, which indicated the loss function of brown AT.Then the exposure dose-dependent and time-dependent trend of adipokine levels of m RNA and protein were assessed by RT-q PCR and ELISA. Results showed that there was upregulation among pro-inflammation cytokines, downregulation among anti-inflammation cytokines, and influence among regulators of adipocyte differentiation, appetite and metabolism. And the time-dependent trends of adipokine content were similar both in subcutaneous AT and serum. It illustrated the synchronization of inflammation both in adipose tissue and serum, and suggested that adipose tissue play an important regulating effect of inflammation and metabolic, except the functioning of energy supply.