• YAKHAK HOEJI
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• v.40 no.4
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• pp.442-448
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• 1996

Mitomycin C(MMC) has been used as an anticancer drug and behaves as an alkylating agent forming covalent cross-link between complementary strands of double strand DNA. The purpose of this research was to determine number of DNA adducts, formed in vivo by Mitomycin C, in mouse organs. DNAs from liver, lung, brain and pancreas were isolated and used for $^{32}P$-postlabelling. The labeled nucleotides were separated by 2D-TLC and subjected to autoradiography. Numbers of MMC-DNA adducts were 9,9,5,4 in liver, pancreas, lung and brain, respectively.

• Biomolecules & Therapeutics
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• v.1 no.1
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• pp.58-64
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• 1993

DNA addicts induced by putative chemical related to carcinogenesis were detected and determined by $^{32}$P-Postlabelling assay after exposure of 4 compounds comprising two auto dyes (amaranth, new coccine) and two flavonoid compounds (rutin, quercetin) to ICR mouse. DNA was isolated from mouse liver and digested enzymatically to deoxyribonucleoside 3'-monophosphate. The postincubation of DNA digests with nuclease Pl before $^{32}$P-labelling enhanced the technique's sensitivity. Nuclease Pl cleaves deoxyribonucleoside 3'-mono-phosphates of normal nucleotides to deoxyrihonucleosides which do not serve as substrates for polynucleotide kinase, while most of addicts were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease Pl. The adducted deoxyribonucleoside 3'-monophosphate was converted to 5'-$^{32}$P-labelled deoxynucleoside 3',5'-bisphosphate by T4 polynucleotide kinase. The nucleotides were separated by anion-exchange thin layer chromatography(TLC) on polyethyleneimine cellulose by 4-dimensional or 2-dimensional TLC then detected by autoradiography. The results show that DNA addicts were detected in liver DNA of ICR mouse after administration of amaranth and quercetin by 2-dimensional and/or 4-dimensional TLC.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.10 no.1
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• pp.1-17
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• 2000

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 nucleophilic 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 $^{32}P$-postlabelling method as one among them. A major project for biomonitoring workers with carcinogen-DNA adducts is to develop non-invasive 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 $^{32}P$-postlabelling for calculating RAL. $[{\gamma}-^{32}P]ATP$ using for $^{32}P$-postlabelling, can synthesize with $[^{32}P]H_3PO_4$, 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 $89.0{\times}10^7$ and $57.0{\times}10^7$ in them. In conclusion, we can significantly measure the DNA adduct in exfoliated urothelial cells by using the above $^{32}P$-postlabelling procedures, and use them to be biomonitoring workers who exposed carcinogens.

• Environmental Analysis Health and Toxicology
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• v.9 no.1_2
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• pp.37-51
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• 1994

To evaluate the genotoxicities of workers exposed to glue and glue cleaning solution, ambient air monitoring of working place, animal study and human monitoring were carried out. By GC-MS analysis, air samples collected from shoesmaking plant were found to be toluene, xylene, cyclohexane, n-hexane, methyl ethyl ketone, trichloroethylene, butylacetate, isopropyl alcohol. Glue and glue cleaning solution from shoesmaking plant were applicated topically to the CD-1 mice. DNA was isolated from skin 24 hr following the application and analysed for DNA-adducts using the nuclease $P_1$version of $^{32}$P-postlabelling assay. RAL (Relative Adduct Labelling, adducts$10^8$ nucleotides) was significantly increased in a dose-dependent manner in the glue cleaning solution treated mice skin. Peripheral blood DNA-adducts of workers exposed to glue and glue cleaning solution were also analysed by the same method, but there were not significant differences in the peripheral blood DNA-adducts level between exposed and control workers. In addition, glue cleaning solution from shoes factory was evaluated for mutagenicity in the Salmonella plate incorporation assay using strains TA 100 and TA 1535 in the presence and absence of Arochlor 1254-induced rat liver S$_{9}$. There was evident mutagenicity for cleaning solution in TA 100 regardless of $S_9$, but TA 1535 showed positive only in the absence of $S_9$when predicted by Stead model of mutagenicity prediction (p=0.0000). The urine concentrates from workers and controls were also assayed for mutagenicity towards strain TA 100 of Salmonella typhimurium in the presence of $S_9$ using Kado's microsuspension assay, but their mutagenic activities were not found to be significant. These data suggest that shoesmaking workers are exposed to genotoxic compounds and need to be monitored by testing the mutagenicity of human urines. However, $^{32}$P-postlabelling application requires further validation for the routine monitoring of human exposure.osure.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.41-42
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• 2001

The chemopreventive effect of tea against 2-amino-l-methyl-6-phenylimidazo［4, 5-b ］pyridine (PhIP)-DNA adduct formation and its mechanism were studied. Rats were exposed to freshly prepared aqueous extracts of green tea (3% w/v) as the sole source of drinking water for 10 days prior to administration with a single dose of PhIP (10 mg/kg body wt) by oral gavage. PhIP-DNA adducts in the liver, colon, heart, and lung were measured using the $^{32}$ P-postlabelling technique.(omitted)