• Proceedings of the Korean Society of Toxicology Conference
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• 2002.05b
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• pp.1-19
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• 2002

It is well established that the initiating event in chemical carcinogenesis is the binding of reactive carcinogens to DNA. Thus, a number of analytic methods have been developed for determining levels of carcinogen-DNA adducts in humans as a marker of individual exposure and, potentially, of risk for cancer development. In addition, reactive carcinogens also bind to protein suggesting protein adducts can be used as a surrogate for DNA adducts in some situations. We have developed monoclonal and polyclonal antibodies to carcinogen-DNA and protein adductis and highly sensitive ELISA and immunohistochemical assays for determining levels of adducts in human tissues. These studies have demonstrated higher levels of adducts in those with higher exposure as a result of workplace, dietary, chemotherapy, environmental of lifestyle (smoking) exposures. Elevated levels of adducts have been found in lung and liver cancer cases compared to controls. We have also used DNA adducts to determine efficacy of an antiosidant vitamin intervention. DNA adduct studies have demonstrated very different levels of damage in those with similar exposure levels. These interindividual differences are likely the result genetic differences in capacity to activate carcinogens, detoxify reactive intermediates and repair DNA adducts once formed. We are currently investigating the relationship between polymorphisms in a number of these genes to determine their relationship to adduct levels as well as their ability to confer increased risk for cancer development. The ability to identify high risk individuals will allow the targeting of screening and preventive strategies to those most likely to benfit.

• Toxicological Research
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• v.14 no.4
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• pp.525-533
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• 1998

The covalent interactions of toluenediisocyanate (TDI) with macromolecules were investigated both in vitro and in vivo. In vitro incubations of 2,4- and 2,6-TDI with DNA or proteins resulted in dose-dependent formation of TDI-protein and TDI-DNA adducts. TDI-treated DNA was highly resistant to enzymatic digestion and thermal hydrolysis, but was readily hydrolyzed under acidic conditions by releasing its corresponding toluenediamine (TDA), suggesting that TDI caused the crosslinking of DNA. Reaction of TDI with albumin and globin resulted in the formation of several adducts, and some adducts were formed in blood of TDI-treated rats in a dose-dependent fashion. Administration of TDI to rats resulted also in a dose-dependent binding of TDI to hepatic tissue. Levels of TDI-albumin adducts were 10 times higher than those of TDI-globin adducts; the biological half lives of TDI-albumin and TDI-globin adducts were 1.2 and 12.5 days, respectively. Globin adducts were detected up to 28 days after the treatment. Hepatic TDI protein adducts were persistent for a substantial period whereas the levels of hepatic TDI-DNA adduct were decreased rapidly. These results indicate that the isocyanato group of TDI is not readily hydrolyzed under physiological conditions, is transported to other organs, and is bound to DNA and/or proteins without further metabolic activation. As the adducted products degrade in the body, TDA is released and introduced to the liver. TDA may additionally bind to hepatic tissue after metabolic activation. Thus, the toxic effect of TDI exposure is considered to persist during the lifetime of the adducted biological macromolecules.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.8 no.2
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• pp.289-295
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• 1998

Used gasoline engine oils(UGEO) are carcinogenic in long term studies and capable of increasing the number of carcinogen-DNA adducts in short term studies when dermally applied to mice. The carcinogenic risk of UGEO has been attributed to the concentration of polycyclic aromatic hydrocarbons(PAH) which accumulate in the lubricating system during the combustion of gasoline. When dermally exposed to UGEO, the use of hand cleanser was commonly recommended for removing it. But generally workers who dermally exposed oils, use kerosene as cleaner which make skin trouble. During this study, female mice aged 4-6 weeks were utilized to evaluate the efficiency of kerosene, as solvent-based cleanser, following dermal exposure to UGEO. DNA adduct were detected at skin and lung tissues by using the $^{32}P$-postlabeling method. Washing with cleansers were done at two different interval times following dermal application of UGEO. The total DNA adducts in skin and lung tissues were statistically significantly increased in positive control groups, and of which the total adduct level in skin tissues was statistically significant higher than those in lung tissues(p=0.005). When washing kerosene, the DNA adduct level in skin tissues was statistically significantly decreased(p=0.0001). But DNA adducts in lung tissue was statistically increased(p=0.0039), and that washed at 8hr post exposure was more severly increase(p<0.05). The slope of regression between DNA adducts of lung between skin tissues was 1.0802. In conclusion, skin cleaning with kerosene facilitates passage of carcinogens to the lungs of animals dermally treated with used gasoline engine oils(UGEO).

• 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.

• 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.

• BMB Reports
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• v.36 no.1
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• pp.1-11
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• 2003

The causative role of polycyclic aromatic hydrocarbons (PAH) in human carcinogenesis is undisputed. Measurements of PAH-DNA adduct levels in easily accessible white blood cells therefore represent useful early endpoints in exposure intervention of chemoprevention studies. The successful applicability of DNA adducts as early endpoints depends on several criteria:i.adduct levels in easily accessible surrogate tissues should reflect adduct levels in target-tissues, ii. toxicokinetics and the temporal relevance should be properly defined.iii. sources of inter- and intra-individual variability must be known and controllable, and finally iv. adduct analyses must have advantages as compared to other markers of PAH-exposure. In general, higher DNA adduct levels or a higher proportion of subjects with detectable DNA adduct levels were found in exposed individuals as compared with non-exposed subjects, but saturation may occur at high exposures. Furthermore, DNA adduct levels varied according to changes in exposure, for example smoking cessation resulted in lower DNA adduct levels and adduct levels paralleled seasonal variations of air-pollution. Intra-individual variation during continuous exposure was low over a short period of time (weeks), but varied significantly when longer time periods (months) were investigated. Inter-individual variation is currently only partly explained by genetic polymorphisms in genes involved in PAH-metabolism and deserves further investigation. DNA adduct measurement may have three advantages over traditional exposure assessment: i. they can smooth the extreme variability in exposure which is typical for environmental toxicants and may integrate exposure over a longer period of time. Therefore, DNA adduct assessment may reduce the monitoring effort. ii. Biological monitoring of DNA adducts accounts for all exposure routes. iii. DNA adducts may account for inter-individual differences in uptake, elimination, distribution, metabolism and repair amongst exposed individuals. In conclusion, there is now a sufficiently large scientific basis to justify the application of DNA adduct measurement as biomarkers in exposure assessment and intervention studies. Their use in risk-assessment, however, requires further investigation.

• Toxicological Research
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• v.33 no.4
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• pp.265-272
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• 2017

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the $N^2$ or C8 positions of guanine or the $N^6$ position of adenine. The proportion of $N^2$ and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases-Pol ${\eta}$, ${\kappa}$, ${\iota}$, and ${\zeta}$ and Rev1-in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2'-deoxyguanosin-$N^2$-yl)-3-aminobenzanthrone (dG-$N^2$-ABA) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-$N^2$-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-$N^2$-ABA. dG-$N^2$-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol ${\eta}$ and ${\kappa}$ are stronger contributors to TLS over dG-C8-ABA, and Pol ${\kappa}$ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.290.1-290.1
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• 2003

Oxidative DNA damage has been associated with many disease. Quantation of DNA adducts is considered to be a useful biomarker of oxidative DNA damage because its formation can also be induced by oxidative stress. Extensive efforts have been taken to identify the analytical methods for minimizing the artifactual formation of oxidative DNA damage. We have done direct analysis of DNA adducts using LC/ESI-MS without urine sample extraction. (omitted)

• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.263-269
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• 1998

It is an established fact that most of the carcinogens implicate bay-region diol epoxides as the ultimate carcinogenic metabolites. These electrophiles react with nucleophilic sites in the cells to form abducts. It is the formation of carcinogenic-DNA adducts that is thought to initiate carcinogenesis. In our previous study we have reported chemopreventive property of Ginseng on 7,12-dimethylbenz (a)anthracene (DMBA) induced skin papillomagenesis in male Swiss albino mice. In this study we have examined the effect on formation of DMBA-DNA adducts in rat hepatocytes pretreated with ginseng. Primary cultures of rat hepatocytes were used. The cells wets treated with ginseng for 24 hrs and then with DMBA (iOn) for 18 hrs. Cells were then harvested, their DNA was isolated and analyzed by P)2 labelling. A significant reduction in the levels of DMBA-DNA adduces (adducts/108 nucleotides) was observed in all cultures pretreated with ginseng. The viability of cells was not affected by pre-treatment with ginseng. Our finding suggests that ginseng block or suppresses the events associated with chemical carcinogenesis by inhibiting metabolic activation of the carcinogens.

• Environmental Mutagens and Carcinogens
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• v.22 no.4
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• pp.303-311
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• 2002

This study evaluated the concentrations of urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) in industrial waste incineration workers. The effect of genetic polymorphisms of xenobiotic metabolism enzymes on urinary concentration of PAH metabolites was assessed. And, aromatic DNA adduct levels were also determined in total white blood cells. Fifty employees were recruited from a company handling industrial wastes located in Ansan, Korea: non-exposed group (n=21), exposed group (n=29). Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects near incinerators. Urinary 1-hydroxypyrene glucuronide (1-OHPG), a major pyrene metabolite, was assayed by synchronous fluorescence spectroscopy after immunoaffinity purification using monoclonal antibody 8E11 (SFS/IAC). Multiplex PCR was used for genotyping for GSTMI/TI and PCR-RFLP for genotyping of CYP1A1 (MspI and Ile/Val). PAH-DNA adducts in peripheral blood WBC were measured by the nuclease P1-enhanced postlabeling assay. Smoking habit, demographic and occupational information were collected by self-administered questionnaire. The range of total ambient PAH levels were 0.00-7.00 mg/㎥ (mean 3.31). Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (p=0.006, by Kruskal-Wallis test). There was a statistically significant dose-response increase in 1-OHPG levels with the number of cigarettes consumed per day (Pearson correlation coefficient=0.686, p<0.001). Urinary 1-OHPG levels in occupationally exposed smoking workers were highest compared with non-occupationally exposed smokers (p=0.053, by Kruskal-Wallis test). Smoking and GSTMI genotype were significant predictors for log-transformed 1-OHPG by multiple regression analysis (overall model R²=0.565, p<0.001), whereas smoking was the only significant predictor for log-transformed aromatic DNA adducts (overall model R²=0.249, p=0.201). Aromatic DNA adducts was also a significantly correlation between log transferred urinary 1-OHPG levels (pearson's correlation coefficient=0.307, p=0.04). However, the partial correlation coefficient adjusting for Age, Sex, and cigarette consumption was not significant (r=0.154, p=0.169). The significant association exists only in individuals with the GSTMI null genotype (pearsons correlation coefficient=0.516, p=0.010; partial correlation coefficient adjusting for age, sex, and cigarette consumption, r=0.363, p=0.038). Our results suggest that the significant increase in urinary 1-OHPG in the exposed workers is due to higher prevalence of smokers among them, and that the association between urinary PAH metabolites and aromatic DNA adducts in workers of industrial waste handling may be modulated by GSTMI genotype. There results remain to be confirmed in future larger studies.