• Journal of Environmental Health Sciences
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• v.40 no.1
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• pp.55-62
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• 2014

Objectives: $^{32}P$-postlabeling assay is the most sensitive method of detecting DNA adduct formation. However, it is limited by a low sample throughput and use of radioisotopes (RI). In this study, we modified it to minimize these limitations and applied it to Z. platypus exposed to Benzo(a)pyrene (BaP) in order to investigate DNA adduct formation (effect biomarker for pollutants) in Z. platypus for assessing risk of waterborne BaP exposure. Methods: DNA hydrolysis was performed only with Micrococcal nuclease (MNase), RI reduction test was performed and the overlapping steps between thin layer chromatography (TLC) and radioisotope high-performance liquid chromatography (RI-HPLC) were omitted. The application of a modified method to Z. platypus exposed to BaP was performed. Results: The results revealed that the amount of RIs used can be reduced roughly 10-fold. Because the analysis time was shortened by 8.5 hours, the sample throughput per hour was increased compared with the previous method. The results of applying modified $^{32}P$-postlabeling assay to Z. platypus, DNA adduct formation in Z. platypus showed dose-dependency with the BaP concentration. Only BPDE-dGMP was detected as a DNA adduct. Conclusion: These results demonstrate that the modified $^{32}P$-postlabeling assay is a suitable method for detecting DNA adduct formation in Z. platypus exposed to waterborne BaP and will be useful in risk assessment of carcinogenic effect in aquatic environment due to BaP.

• Journal of Environmental Health Sciences
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• v.28 no.1
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• pp.21-29
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• 2002

To identify and evaluate the dichlorobenzidine(DCB)-DNA adducts in liver cell and bladder epithelial cells by $^{32}$ P-postlabeling and GC/MS-SIM, we orally exposed the dichlorobenzidine(20mg/kh body wt./day) to male Sprague-Dawley rats(l85$\pm$10g) for 14 days. Two kinds of DCB-DNA adduct(A1 and A2) were found at the same site of thin layer chromatogram of $^{32}$ P-postlabeling method in liver cells and bladder epithelial cells. In liver cells, relative adduct labeling(RAL) $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 34.1$\pm$3.71 and 69.9$\pm$5.02, that of adduct A2 were 74.1$\pm$10.1 and 105.1$\pm$10.1 on 10 and 14 days after treatment, respectively. And in bladder epithelia cells, RAL $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 5.92$\pm$1.60 and 15.9$\pm$1.31, that of adduct A2 were 9.81$\pm$2.81 and 22.8$\pm$1.79 on 10 and 14 days after treatment, respectively. DCB metabolites formed DNA adducts were monoacetyl-dichlorobenzidine(acDCB) and diacetyl-dichlorobenzidine(di-acDCB), which was identify by gas chromatography/mass spectrometry-scan ionization mode(GC/MS-SIM), after hydrolysis of DCB-DNA adducts isolated from live cells and bladder epithelial cells. The base peak of acDCB were 252 and 294 m/z, and that of di-acDCB were 252, 294 and 336 m/z. In conclusion, the exposed DCB formed two kinds of DCB-DNA adduct, the proximate materials of that were acDCB and di-acDCB in liver and bladder epithelial cells. And the above GC/MS-SIM method was found the DCB-DNA adducts could be monitoring by gas chromatography.

• Journal of Environmental Health Sciences
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• v.24 no.3
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• pp.92-98
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• 1998

작업장에서 근로자들이 엔진오일 등 각종 오일에 피부가 폭로되었을 때 이것을 쉽게 세척하기 위하여 일반적으로 솔벤트를 사용한다. 그러나 솔벤트를 사용하면 피부를 건조하게 만들 뿐만 아니라 오일에 함유되어 있는 각종 성분들을 피부내에 흡수되는 것을 촉진시킬 수 있어서 이에대한 대처방법이 요구된다. 특히 폐가솔닐엔진오일데는 방향족탄화수소(PAHs)와 같은 물질이 함유되어 있어 체내에 흡수되면 발암물질로 대사되어 표적장기(피부와 폐조직)에서 DNA adducts를 높은 수준으로 형성한다고 알려져 있다. 본 연구에서는 식물기름에서 구할 수 있는 d-라이모닌(Limonene)를 세척제로 사용하여 폐가솔린엔진오일의 폭로로 인하여 형성되는 DNA adducts를 $^{32}P-postlabeling방법으로 분석함으로써 d-라이모닌의 세척효과를 평가하고자 하였다. HDC(ICR) Br 자성마우스의 견갑골 부위에 있는 털을 제거하고 그 부위에 폐가솔린엔진오일을 폭로시키고 1시간과 8시간이 지난 다음에 d-라이모닌으로 각각 세척을 하였다. 마지막 폭로를 마치고 24시간이 지난 다음에 실험동물을 희생시켜 표적장기(폭로된 피부와 폐)에서 시료를 채취하였다. 먼저 시료에서 DNA를 분리하여 가수분해한 다음에 $^{32}P-postlabeling하여 DNA adducts를 분리하였다. 폐가솔린엔진오일만 폭로시킨 그룹의 피부와 폐조직에 형성된 DNA adducts가 각각 30.3$\pm$3.7과 15.7$\pm$2.4로서 대조군(2.5$\pm$1.0과 1.4$\pm$0.4)에 비하여 통계적으로 유의하게 높았고 (p<0.01), 또한 폐조직에서 보다 피부조직에서 통계적으로 유의하게 높았다(p<0.01). 폐가솔린엔진오일을 폭로시킨 후에 d-라이모닌으로 세척한 그룹에서는 피부와 폐조직에 형성된 DNA adducts가 통계적으로 유의하게 감소하였는데(p<0.01), 8시간 보다는 1시간이 지난 다음에 세척한 그룹에서 DNA adducts의 감소현상이 더 크게 나타났다. 결론적으로 피부에 폭로된 폐가솔린 엔진오일을 d-라이모닌으로 세척하면 폐가솔닐엔진오일내에 함유된 발암물질이 체내흡수되는 것이 억제되고, 피부와 폐조직 모두에서 DNA adducts의 형성을 감소시킬 수 있으며, 폐오일이 폭로된 후 빨리 세척하는 것이 더 효과적임을 증명하였다.

• Proceedings of the Korean Environmental Health Society Conference
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• 2002.04a
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• pp.49-51
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• 2002

To identify and evaluate the dichlorobenzidine(DCB)-DNA adducts in liver cell and bladder epithelial cells by $^{32}$ P-postlabeling and GC/MS-SIM, we orally exposed the dichlorobenzidine (20mg/kh body wt.,/day)to male sprague-dawley rats for 14 days. Two kinds of DCB-DNA adduct were found at the same site of thin layer chromatogram of $^{32}$ P-postlabeling method in liver cells and bladder epithelial cells. In liver cells, relative adduct labeling(RAL) $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 34.1$\pm$3.71 and 69.9$\pm$5.02, that of adduct A2 were 74.1$\pm$10.1 and 105.1$\pm$10.1 on 10 and 14 days after treatment, respectively. And in bladder epithelia cells, RAL $\times$ 10$^{12}$ of DCB-DNA adduct A1 were 5.92$\pm$1.60 and 15.9$\pm$1.31, that of adduct A2 were 9,81$\pm$2.81 and 22.8$\pm$1.79 on 10 and 14 days after treatment, respectively. DCB metabolites formed DNA adducts were monoacetyl-dichlorobenzidine(acDCB) and diacety1-dichlorobenzidine(di-acDCB), which was identify by gas chromatography/mass spectrometry-scan ionization mode(GC/MS-SIM), along with hydrolysis, extraction and TFA(trifluoroacetyl anhyride) derivatization with DCB-DNA adducts isolated from live cells and bladder epithelial cells. The base peak of acDCB were 252 and 294 m/z, and that of di-acDCB were 252, 294 and 336 m/z. In conclusion, the exposed DCB formed two kinds of DCB-DNA adduct, the proximate materials of that were acDCB and di-acDCB in liver and bladder epithlial cells. And the above GC/MS-SIM method was found the DCB-DNA adducts could be monitoring by gas chromatography.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.3 no.1
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• pp.14-21
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• 1993

N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NAH are found in significant amounts in tobacco condensates, synthetic fuels, polluted river sediment, and effluents from the heating of coal. Following topical application 7H-dibenzo[c, g]carbazole (DBC) induces cancer in liver as well as skin, indicating that dermal exposure can lead to systemic effect. DBC and dibenz[a,j]acridine (DBA) are examples of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBC and DBA's, DBA, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. There were six adducts that were related to DBC application. These addusts were seen in the target organ, liver at high levels, but at very low levels in non-target organs, skin, lung and kidney. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD higher than that of parent compound. Two adducts were seen when DBA-5,6-DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD.

• 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.

• Toxicological Research
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• v.17
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• pp.139-144
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• 2001

Chewing betel quid is associated with an increased risk of oral cancer. The betel quid chewed in Taiwan includes the inflorescence of Piper betle, which contains high concentrations of safrole (15 mg/fresh weight). Piper betle leaf is also used in betel quid; however, the concentration of safrole in betel leaf has not been documented. Chewing betel quid may contribute to safrole exposure in man (420 mm in saliva). Using $a^{32}$P-postlabeling method, we have recently demonstrated the presence of stable safrole-like DNA adducts in human oral tissues following betel quid chewing. Safrole is a rodent hepatocar-cinogen, and the real nature of safrole-DNA adducts in human tissues beside oral has not been elucidated. In this paper, we tested the safrole DNA adducts forming potential in human hepatic and oral derived cells by the ${32}^P$-postlabeling technique. The results suggest that oral cancer derived cell OC-2 alone is not able to form safrole-DNA adduct. However, safrole DNA adducts can be detected following I'-hydroxysafrole, a proximate safrole metabolite, treatment. In addition, pretreament of cytochrome P450 inducers also enhanced the formation of previously undetectable safrole DNA adducts. This finding couples with our previous results suggest that oral may serve as a target tissue for safrole, and safrole may be involved in oral carcinogenesis.

• Journal of Environmental Health Sciences
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• v.29 no.4
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• pp.21-26
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• 2003

We examine the metabolites(DCB and acetyl DCB) extracted from exfoliated urothelial cells of 33 workers who employed DCB-handling industries. The characteristics of workers submitted urine, whose age, working years and smoking persons were 41.9$\pm$11.1, 8.7$\pm$5.5 and 25(32.0%), respectively. DNA adduct was isolated from the exfoliated urothelial cells by applying $^{32}$ p-postlabeling procedure. Metabolites(DCB and acetyl DCB) were extracted from DNA adducts by hydrolyzing and N-glycosylase. Concentrations of DCB and acetyl DCB were 28.6$\pm$5.25 ng/g DNA, and 17.0$\pm$3.73 ng/g DNA, respectively. The regression between DCB level and exposure years of workers is y = 1.668 + 2.588x(p = 0.005, $r^2$= 0.394). The regression between acetyl DCB level and exposure years of workers is y = 8.071 + 1.325x(p = 0.076, $r^2$= 0.222). Smoking workers are significantly higher than non-smoking workers on DCB and acetyl DCB level(p = 0.065 and 0.021, respectively). DCB level was 33.9$\pm$7.14 ng/g DNA on smokers, and 23.1$\pm$9.97 ng/g DNA on non-smokers. Acetyl DCB was 25.1$\pm$5.27 ng/g DNA on smokers, and 8.92$\pm$7.22 ng/g DNA on non-smokers.

• Toxicological Research
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• v.6 no.1
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• pp.63-73
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• 1990

Human exposure to environmental carcinogens can be detected by a number of methods including immunoassay, $^{32}P-postlabeling$ assay, and fluorescence technique. These assays have been applied to measure biological markers of carcinogen-adducts formed with macromolecules such as DNA, RNA and protein. In an attempt to investigate causal relationships between carcinogen exposure and tumor formation, specific carcinogen-adducts have been quantitated from human tissues and body fluids of cancer patients, occupational workers heavily exposed to certain carcinogens, smokers and controls. Carcinogens studied for biological human monitoring include benzo(a)pyrene, aflatoxin B1, UV light, ethylene oxide, 8-methoxypsoralen, 4-aminobiphenyl, vinyl choride, N-nitrosamine, cisplatin and other chemotherapeutic agents. Relevance of human monitoring for cancer research, progress in this field, methods to detect carcinogen-adducts are reviewed here. It is hoped that these approaches will be used for the risk assessment of carcinogen exposure, cancer etiology study and cancer prevention in humans.

• Toxicological Research
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• v.6 no.1
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• pp.61-61
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• 1990

Human exposure to environmental carcinogens can be detected by a number of methods including immunoassay, $^{32}P$-postlabeling assay, and fluorescence technique. These assays have been applied to measure biological markers of carcinogen-adducts formed with macromolecules such as DNA, RNA and protein. In an attempt to investigate causal relation ships between carcinogen exposure and tumor formation, specific carcinogen-adducts have been quantitated from human tissues and body fluids of cancer patients, occupational workers heavily exposed to certain carcinogens, smokers and controls. Carcinogens studied for biological human monitoring include benzo(a)pyrene, aflatoxin B1, UV light, ethylene oxide, 8-methoxypsoralen, 4-aminobiphenyl, vinyl chloride, N-nitrosamine, cisplatin and other chemotherapeutic agents. Relevance of human monitoring for cancer research, progress in this field, methods to detect carcinogen-adducts are reviewed here. It is hoped that these approaches will be used for the risk assessment of carcinogen exposure, cancer etiology study and cancer prevention in humans.