• 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 Ginseng Research
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• v.13 no.1
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• pp.42-48
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• 1989

The binding of bay region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs) to target tissue DNA is thought to be essential for the initiation of cancer by these compounds. In this study we investigated the effect of polyacetylenes such as panaxynol and panaxydol on the formation of benzo(a)pyreno (BP)-metabolite-DNA adduct in the liver of ICR mice. Treatment of mice by i.p. administration of polyacetylenes produced a marked reduction in BP metabolite binding to DNA in vitro. Following i.v. administration of (3H)BP(300, ${\mu}$Ci/21 nmoles/0.1 nt DMSO) to mice, radioactivity was detected in the DNA of the liver in vivo. The result of tentative identification of the 4 peaks between the two standard markers for high pressure liquid chromatography showed that the peaks. I, II, III, and IV were BP-phenol oxide-DNA adduct (or BP-diol-epoxide-dCyt. adduct), (-) BP$.$diolepoxide I:dGuO adduct, (+) BP-diol-epoxide I: dGuo adduct, and BP-diol-epoxide II:dGuO adduct, respectively. The minor adduct, (-) BP-diol epoxide I: dGuo was reduced to 6971 of the amount of the control, while the major adduct, (+) BP-diolepoxide I: dGuO(peak II) which was produced from (-) BP-7, 8-diol was reduced to 78% of that of the control. The amount of the minor adduct, BP-diol-epoxide II:dGuo adduct(peak IV) which formed from (+) BP-7, 8-diol was 58% of the control. These results show that the panaxydol is more related to inhibition of the formation of the minor ad- ducts than of the major adducts, which were generally produced from ($\pm$) BP-7, 8-dihydro-dials.

• 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 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.

• Bulletin of the Korean Chemical Society
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• v.30 no.9
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• pp.2125-2128
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• 2009

• Environmental Mutagens and Carcinogens
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• v.23 no.3
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• pp.99-102
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• 2003

IARC classified areca quid as a human carcinogen. Areca quid chewed in Taiwan includes Piper betle inflorescence, which contains high concentrations of safrole (15 mg/fresh weight). Safrole is a documented rodent hepatocarcinogen, and chewing areca quid may contribute to human exposure (420 $\mu$m in saliva). The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. Using human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s, CYP2E1 and CYP2C9 were identified as the main P450s involved in the activation of safrole. We have demonstrated the presence of stable safrole-dGMP adducts in human oral tissues following areca quid chewing using $^{32}$ P-postlabeling and HPLC mass spectrometry methods. By studying 88 subjects with a known AQ chewing history and 161 matched controls, we have demonstrated that the presence of safrole-DNA adducts in peripheral blood cells was correlated to AQ chewing, and CYP2E1 seemed to play an important role in the modulation of safrole-DNA adduct formation. We have also shown that safrole can form stable safrole-DNA adducts as well as oxidative damages in rodent liver. However, the stable safrole-DNA adducts may represent a more significant initial lesion as compared to the rapidly repaired safrole-induced 8-hydroxy-2'-deoxyguanosine. This oxidative DNA damage is mediated through the formation of hydoryxchavicol, the major safrole metabolite in human urine. Hydroxychavicol may have gone through two-electron oxidation to the o-quinone; then via one-electron reduction to semiquinone radicals to generate oxidative DNA damage. However, these reactive metabolites can be efficiently conjugated by GSH. These data suggest that safrole may contribute to the initiation of oral carcinogenesis through safrole-DNA adduct and not oxidative DNA damage. In addition, CYP2E1 may modulate this adduct formation.

• Korean Journal of Pharmacognosy
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• v.34 no.4 s.135
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• pp.297-302
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• 2003

Cnidii Rhizoma water extract (CRW) was tested for liver cancer chemopreventive potential by measuring the inhibition of phase I enzyme and benzo[a]pyrene-DNA adduct formation and induction of phase II detoxification enzymes. There was 17.0% inhibition in the activity of cytochrome P450 1A1 enzyme with the treatment of 150 mg/ml CRW. At concentration of 30 mg/ml CRW, the binding of $[^3H]B[a]P$ metablites to DNA of NCTC-clone 1469 cell was inhibited by 33.3%. CRW was potent inducer of quinone reductase (QR) and glutathione S-transferase (GST) activities in cultured murine hepatoma Hepalc1c7 cells. However, hepatic glutathione (GSH) level was not influenced by CRW. These findings suggest that CRW has chemopreventive potential of liver cancer by inhibiting cytochrome P450 1A1 activity and benzo[a]pyrene-DNA adduct formation and inducing QR and GST activities.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.05b
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• pp.20-45
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• 2002

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Spragu-Dawley rats. human serum, and standard major lipids were in vitro reacted with benzo[a]pyrene(BP) and BP metabolites. 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(BPDE-I), an ultimate carcinogenic form of BP, was covalently bound to triglyceride(TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high performance liquid chromatography(HPLC). TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids(Phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and nonesterified fatty acids(palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other BP metabolites (BP-phenols and -diols) did not react with TG, which TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-respect to its ability to form an adduct with BPDE-I-lipid adduct in vitro between TG and [1,3-3H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with BP(25 mg/rat). Also, obvious correlations between [3H]BP related-biomolecule adducts (DNA, protein) or lipid damage and the BPDE-I-TG adduct were obtained in various tissues of mice i.p. treated with [3H]BP. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA, and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2000.12a
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• pp.74-74
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• 2000

• Food Science and Preservation
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• v.21 no.5
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• pp.747-756
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• 2014

The objective of this study was to examine the effects of vitamin C on the formation of aflatoxin $B_1$ ($AFB_1$)-DNA adduct and $AFB_1$-induing cellular oxidative damage in rat livers treated with radiation and $AFB_1$. Six-week-old male Sprague-Dawley rats were randomly divided into five groups: the control group, the $AFB_1$-treated group, the group treated with $AFB_1$ and vitamin C, the group treated with X-ray and $AFB_1$, and the group treated with X-ray and $AFB_1$ with vitamin C. On the first day of the experiment, only one dose of X-rays was exposed to the entire liver at 1,500 cGy. Next, vitamin C was injected at 10 mg/kg body weight via intraperitoneal injection, followed an hour later by the administration of 0.4 mg/kg of $AFB_1$ via intraperitoneal injection. These treatments were administered every three days for 15 days. On the 16th day, the animals were sacrificed. The $AFB_1$ contents of the rat sera were determined via indirect competitive ELISA. In the quantitative analysis of $AFB_1$ in the rat sera via ELISA, $5.17{\pm}0.34ng/mL$ of $AFB_1$ was detected in the $AFB_1$-treated groups, but the amount decreased more significantly to $3.23{\pm}0.76ng/mL$ in the groups treated with $AFB_1$ and vitamin C (p<0.01) than in the $AFB_1$-treated groups. The effect of vitamin C on $AFB_1$-DNA adduct formation was determined via ELISA. The values of $AFB_1$-DNA adduct formation were $9.38{\pm}0.41ng/mL$ in the $AFB_1$-treated groups, but the amount decreased more significantly to $5.28{\pm}0.32ng/mL$ in the groups treated with $AFB_1$ and vitamin C (p<0.01) than in the $AFB_1$-treated groups. Immunohistochemistry revealed that the accumulation of the $AFB_1$ was not observed in the normal liver tissue (G1). The $AFB_1$-positive materials were observed in the central vein and the portal vein of the liver tissue from the $AFB_1$(G2) treatment or the X-ray and $AFB_1$(G4) co-treatment, but the $AFB_1$-positive materials were observed weakly in the group treated with vitamin C (G3 and G5). These results indicate that vitamin C had ameliorating effects on the $AFB_1$ accumulation of liver tissue.