• 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 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.

• 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.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.139-146
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• 2000

7- Bromomethylbenz[a]anthracene is a known mutagen and carcinogen. The two major DNA adducts produced by this carcinogen, i.e., $N^2$-(benz[a]anthracen-7-yl methyl)-2'-deoxyguanosine (2, b[a]$a^2$G) and $N^6$-(benz[a]anthracen-7-ylmethyl)-2'-deoxyadenosine (4, b[a]$a^6$/A), as wel 1 as the simpler benzylated analogs,$N^2$-benzyl-2'deoxyguanosine (1, $bn^2$G) and $N^6$-benzyl-2'-deoxyadenosine (3, $bn^6$/A), were prepared by direct aralkylation of 2'-deoxyguanosine and 2'-deoxyadenosine. To determine the site-specific mutagenicity of these bulky exocyclic amino-substituted adducts, the suitably protected nucleosides were incorporated into 16-base oligodeoxyribonucleotides in place of a normal guanine or adenine residues which respectively are part of the ATG initiation codon for the lac Z' \alpha-complementation gene by using an in situ activation approach and automated phosphite triester synthetic methods. The base composition and the incorporation of the bulky adducts into synthetic oligonucleotides were characterized after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis.

• 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.

• 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.

• Toxicological Research
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• v.12 no.1
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• pp.21-27
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• 1996

Vitamin C has been well known to be a potential chemopreventive agent for several toxic compounds. It reduced the mutation frequencies of 6-sulfooxymethylbenzo[a]pyrene (SMBP) and 6hydroxymethylbenzo[a]pyrene (HMBP) in Salmonella typhimurium TA98 and TA100, indicating that corbic acid affects both frameshift and base-pair substitution mtltations. A similar type of dose-response relationship was shown in the V79 cells, although the inhibitory effect was less dramatic compared with that in S. typhimurium. However, SMBP or HMBP binding to calf thymus DNA was not affected by the presence of vitamin C, suggesting that SMBP seems to be much more reactive to calf thymus DNA than vitamin C. This was supported by migration pattern and fluorescence intensity of SMBP-modified plasmid on the gel. These restilts were not correlated with mutation tests in bacterial and mammalian cell systems. It has been already reported that vitamin C inactivates SMBP through the formation of covalently bound addact. It was found from HPLC analysis that the reaction between vitamin C and SMBP was accomplished within just 5 min and then produced the several products. These findings indicate that the beneficiary of vitamin C is not merely derived from the covalent adducts. On the other hand, the addition of DNA to incubation mixture reduced the amounts of vitamin C adducts while the magnitude of HMBP peak increased, suggesting that DNA accelerates the SMBP hydrolysis to intercept the interaction between SMBP and vitamin C or forms rapidly complex with SMBP.

• 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.

• 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.