근로자의 뇨중 상피세포에서 32p-postlabeling에 의한 발암물질의 DNA adduct 측정방법에 대한 연구

Abstract

Carcinogen-DNA adduct analysis has potential for biomonitoring the earliest effects of exposure to many chemical carcinogens. They are the covalent reaction products of electrophiles and nucle ophilic sites on DNA and the initial damage to DNA induced by many carcinogens. So many researchers begin to use them as biomarker for monitoring the earliest exposure of carcinogens and develop the effective analytical techniques about them. Randerath, Gupta and coworkers(1981, 1982) has also developed a 32P-postlabelling method as one among them. A major project for biomonitoring workers with carcinogen-DNA adducts is to develop noninvasive samples instead of tissues of target organs such as baldder and lung. This study use the exfoliated urothelial cells in urine for examine benzidine-DNA adducts. The content of exfoliated urothelial cells is not enough to significantly measure DNA content with spectrophotometer, and require the another way. So firstly washing the collected cells with PBS and 70% ethanol and centrifuge them for removing the crystals in urine, which block the isolation of DNA adducts. And then, measure the total nucleotide after 32P-postlabelling for calculating RAL. [γ-32 P]ATP using for 32P-postlabelling, can synthesize with [32P]H3PO4, and reagent and enzyme mixture (RM, EM), which is very economic in case of requiring a lot of them. Chromatography was composed of two steps. First step was to separate adduct ones from unadducted nucleotide, and secondary step was separate each adduct, which were performed with 4 kinds of solvents and different directions on TLC. With this procedure, we measure the DNA adducts in exfoliated urothelial cells of workers who were employed in benzidine and benzidine- dye company. RAL of adducts were 89.0×107 and 57.0×107 in them. In conclusion, we can significantly measure the DNA adduct in exfoliated urothelial cells by using the above 32P-postlabelling procedures, and use them to be biomonitoring workers who exposed carcinogens.