INVESTIGATION ON TOXICITIES OF ALTERNATIVES FOR CHLOROFLUOROCARBONS AND ITS RISK ASSESSMENT USING MOLECULAR AND BIOLOGICAL MARKERS.

从暴露于1-溴丙烷的大鼠中收集尿液。取尿液100 μL，加入400μL饱和氯化钠溶液，用500μL乙酸乙酯萃取3次。将收集的乙酸乙酯层用干燥的亚砜钠脱水，并用无水蒸发器干燥。用40μL乙酸乙酯分离干燥样品，并用TBDMS衍生化。结果表明，代谢产物N-乙酰-S-丙基半胱氨酸具有剂量依赖性，可作为内暴露标志物。用丙酮-草酸混合液从血样中提取珠蛋白。在氮气保护下，用酸水解珠蛋白，LC-MS/MS鉴定S-丙基半胱氨酸。在深度麻醉下，取1-溴丙烷染毒大鼠大脑、小脑、脑干和脊髓。将组织均质化并获得可溶性级分。采用夹心酶免疫分析法定量测定神经特异性蛋白γ-烯醇化酶和胶质细胞特异性蛋白β-S100。用酶循环法测定谷胱甘肽（GSH）和GSSG。结果表明：1-溴丙烷可引起大鼠大脑γ-烯醇化酶的特异性降低，对中枢神经系统有明显的不良影响。α-烯醇化酶免疫组化染色显示α-烯醇化酶在睾丸支持细胞上有明显的特异性定位。酶免疫定量分析显示睾丸中α-烯醇化酶呈剂量依赖性增加，表明1-溴丙烷对支持细胞的影响。

Urine was collected from the rats exposed to 1-bromopropane. One hundred μL of urine was added 400μL of saturated sodium chloride solution, and then extracted with 500μL of ethyl acetate for three times. The collected ethyl acetate layer was dehydrated with dried sodium sulfoxide, and dried with centrifuging evaporator. The dried sample was resolved with 40μL of ethylacetate and derivatized with TBDMS. The result showed the metabolite N-acetyl-S-propylcysteine was dose-dependent and available as an internal exposure marker.Blood was collected from the rats exposed to 1-bromopropane. Globin was extracted from blood samples using acetone-oxalic acid mixture. Globin was hydrolyzed with acid under nitrogen, and S-propylcysteine was identified using LC-MS/MS.The cerebrum, cerebellum, brainstem and spinal cord were taken from the rats exposed to 1-bromopropane under deep anesthesia. The tissue was homogenized and the soluble fraction was obtained. Neuro-specific protein γ-enolase and glia-specific protein β-S100 was quantified with a sandwich type enzyme immunoassay. Glutatione (GSH and GSSG) was also measured by enzymatic cycling method. The result showed that gamma-enolase was decreased specifically in the cerebrum and 1-bromopropane has a great adverse effect on the central nervous system.The testes were taken from the rats exposed to 1-bromopropane under deep anesthesia. Immunostaining with α-enolase showed clearly specific localization of α-enoalse to Sertoli cells in the testis. The quantification with enzyme immunoassay showed dose-dependent increase in alpha-enolase in the testis, suggesting the effects of 1-bromopropane on Sertoli cells.