Yong-Dae Kim

Assay of 2-naphthol in human urine by high-performance liquid chromatography

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 micromol/mol creatinine) to 34.19 ng/ml (59.11 micromol/mol creatinine), and the mean+/-standard deviation was 5.08 ng/ml (6.60 micromol/mol creatinine)+/-5.75 ng/ml (9.22 micromol/mol creatinine). The mean+/-standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 micromol/mol creatinine)+/-6.16 ng/ml (10.23 micromol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 micromol/mol creatinine)+/-3.92 ng/ml (7.03 micromol/mol creatinine).

TISSUE-DISTRIBUTION OF ALDEHYDE DEHYDROGENASE 2 AND EFFECTS OF THE ALDH2 GENE- DISRUPTION ON THE EXPRESSION OF ENZYMES INVOLVED IN ALCOHOL METABOLISM

In alcohol metabolism, acetaldehyde, a highly reactive intermediate that may cause cellular and DNA damages, is converted to acetate by mitochondrial aldehyde dehydrogenase ALDH2. Although the majority of ingested alcohol is eliminated in the liver, the first-pass metabolism of ethanol in the upper digestive tract is also important for prevention and management of ethanol-related gastrointestinal diseases. However, the tissue-distribution of Aldh2 in mice has been poorly investigated. In this study, therefore, we investigated the tissue-distribution of Aldh2 as well as Aldh1, Cyp1a1, Cyp2e1, and Cyp4b1 in wild type and Aldh2-null mice by immuno-histochemical analysis. The human liver and esophageal tissues were also examined. In mice, the Aldh2 protein was detected in the liver, lung, heart, kidney, testis, esophagus, stomach, colon, and pancreas, suggesting that the tissue-distribution of Aldh2 in mice is similar to that in humans. Therefore, Aldh2-null mice may be useful model animals for the investigation of alcohol metabolism and related diseases. Compared with the wild type, the expression level of Cyp2e1 was increased in the liver from Aldh2-null mice based on Western blot analysis, whereas the levels of Aldh1, Cyp1a1, and Cyp4b1 were indistinguishable. This observation suggests that a metabolite(s) of Aldh2 might down-regulate the expression of Cyp2e1 gene.

Identification of cytochrome P450 isoforms involved in 1-hydroxylation of pyrene

Urinary 1-hydroxypyrene (1-OHP) has been used as a biomarker for environmental polyaromatic hydrocarbon (PAH) exposure. However, it is known that there is an interindividual variability in metabolism of pyrene to 1-OHP depending on the activities of the metabolizing enzymes, especially cytochrome P450s (CYPs). In this study, we investigated the 1-hydroxylation of pyrene by 10 forms of cDNA-expressed human P450s in order to identify the principal isoforms of P450s that are involved in the major metabolic pathway of pyrene. The pyrene 1-hydroxylation activity was found to be the highest in CYP1A1 at both 0.5 and 50microM of pyrene, followed by CYP1B1 and 1A2, whereas other enzymes, including CYP2A6, 2C8, 2C9*1, 2C19, 2D6, 2E1, 3A4, and control microsomes, showed very low or undetectable rates of 1-hydroxylation. In conclusion, CYP1A1, 1B1, and 1A2 are major metabolizing enzymes in 1-hydroxylation of pyrene in vitro. This suggests that the individual difference of these enzymes must be included in epidemiological studies to evaluate PAH exposure using urinary 1-OHP.

Effects of Genetic Polymorphisms in Metabolic Enzymes on the Relationships between 8-hydroxydeoxyguanosine Levels in Human Leukocytes and Urinary 1-hydroxypyrene and 2-naphthol Concentrations

Effects of Genetic Polymorphisms in Metabolic Enzymes on the Relationships between 8‐hydroxydeoxyguanosine Levels in Human Leukocytes and Urinary 1‐hydroxypyrene and 2‐naphthol Concentrations: Yong‐Dae Kim, et al. Department of Preventive Medicine, College of Medicine, Chungbuk National University, South Korea—This study was designed to investigate the relationship between environmental exposure to polycyclic aromatic hydrocarbons (PAHs) and oxidative stress, and to evaluate the effects of cigarette smoking and the genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, NAT2 and UGT1A6 on the relationship. The subjects of this study were 105 healthy Korean males without occupational exposure to PAHs. The 8‐hydroxydeoxyguanosine (8‐OHdG) level in leukocytes, and urinary 1‐hydroxypyrene (1‐OHP) and 2‐naphthol concentrations, were measured by high‐performance liquid chromatography. Genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, NAT2 and UGT1A6 were identified by PCR and PCR‐RFLP methods. The 8‐OHdG level showed a significant correlation with the 1‐OHP concentration in all subjects (p< .001) and in smokers (p< .01), and with the 2‐naphthol level in nonsmokers (p< .01). The 8‐OHdG level was significantly higher in smoking rapid acetylators than in smoking slow or intermediate acetylators, and in individuals with the UGT1A6 wild‐type than in those with the UGT1A6 mutant genotype. Significant positive correlations between 8‐OHdG and 1‐OHP concentrations were found in subjects with every genotype of the CYP1A1 and CYP2E1 genes, with the GSTM1 null‐type, with the NAT2 genotype of a rapid acetylator, and with the UGT1A6 wild‐type, respectively. The urinary 2‐naphthol level significantly correlated with the 8‐OHdG level only in subjects with the GSTM1 null‐type. In conclusion, there is a significant correlation between the 8‐OHdG level in leukocytes and the urinary 1‐OHP concentration in the population not occupationally exposed to PAHs. This relationship is affected by genetic polymorphisms in PAH metabolic enzymes.

Formaldehyde exposure levels and serum antibodies to formaldehyde-human serum albumin of Korean medical students.

In our study, we estimated formaldehyde exposure levels of Korean medical students during their cadaver dissection practice hours. In addition, we examined the prevalence rates of formaldehyde-specific immunoglobulin E or immunoglobulin G antibodies and compared the results with the symptoms the students experienced as a result of formaldehyde exposure. There were 167 Korean medical students (i.e., subjects) aged 23.8+/-2.5 y (mean+/-standard deviation) and a control group of 67 premedical students aged 20.1+/-2.8 y (mean+/-standard deviation). Concentrations of formaldehyde in the cadaver dissection practice laboratory ranged from 0.194 to 11.245 mg/m3 (3.736+/-3.478 mg/m3 [mean+/-standard deviation]). Students reported by self-administered questionnaires that eye soreness (92.8%) and lacrimation (74.9%) were the most common symptoms they experienced during the laboratory sessions. One (0.6%) of the 167 medical students had a history of wheezing during dissection. Fourteen (8.4%) had specific immunoglobulin G antibody, but none had specific immunoglobulin E antibody. These results suggest that (a) Korean medical students are exposed to formaldehyde at a relatively high levels in their dissection practice hours, (b) specific immunoglobulin G is not related to adverse eye or respiratory symptoms, and (c) specific immunoglobulin E is rarely induced as a result of exposure to formaldehyde.

Effects of Dietary Factors and the NAT2 Acetylator Status on Gastric Cancer in Koreans

Environmental dietary carcinogens and genetic polymorphisms in metabolic enzymes have been reported to be the risk factors for gastric cancer. This study was undertaken to investigate the effects of the diet, the N‐acetyltransferase (NAT) 2 acetylation status and their interaction on gastric cancer risk. The study population consisted of 471 gastric cancer patients and 471 age‐ and sex‐matched control subjects. NAT2 genotypes were identified using single‐nucleotide primer extension reaction methods. Thirty‐one alleles related to 12 polymorphism sites were assayed in this study. Significantly increased odds ratios were observed in former smokers (OR = 2.39, 95% CI = 1.57–3.62), heavy drinkers (OR = 1.28, 95% CI = 1.06–1.55) and individuals who eat well‐done meat (OR = 1.24, 95% CI = 1.09–1.41). The odds ratios (95% CI) for high intake of kimchi, stews and soybean paste were 3.27 (2.44–4.37), 1.96 (1.50–2.58) and 1.63 (1.24–2.14), respectively. The NAT2 genotype alone was not associated with gastric cancer risk. A significant gene–environment interaction was observed between environmental carcinogens and NAT2 genotypes. The odds ratios for kimchi, stews and soybean paste were higher in slow/intermediate acetylators than in rapid acetylators. The odds ratios for slow/intermediate acetylators were 2.28 (95% CI: 1.29–4.04) for light smokers and 3.42 (95% CI: 2.06–5.68) for well‐done meat intake. The NAT2 acetylator genotype may be an important modifier of the effects of environmental factors on gastric cancer risk.

Ethanol-induced oxidative DNA damage and CYP2E1 expression in liver tissue of Aldh2 knockout mice.

Ethanol‐Induced Oxidative DNA Damage and CYP2E1 Expression in Liver Tissue of Aldh2 Knockout Mice: Yong‐Dae Kim, et al. Department of Preventive Medicine and Medical Research Institute, College of Medicine, Chungbuk National University, Republic of Korea—Excessive alcohol consumption is associated with increased risks of many diseases including cancer. We evaluated oxidative DNA damage in Aldh2 +/+ and Aldh2 –/– mice after they had been subjected to acute ethanol exposure. Olive tail moment, which was measured using a comet assay, was not increased by ethanol treatment in both Aldh2 +/+ and Aldh2 –/– mice. However, after controlling for the effect of ethanol exposure, the Aldh2 genotype was a significant determinant for Olive tail moments. Although the ethanol treatment significantly increased the hepatic 8‐OHdG generation in only Aldh2 +/+ mice, the level of 8‐OHdG was the highest in Aldh2 –/– ethanol treated mice. The increase in the level of 8‐OHdG was associated with hepatic expression of cytochrome P450 2E1 (CYP2E1). The levels of Olive tail moment and the hepatic 8‐OHdG in the Aldh2 –/– control group were significantly higher than those of the Aldh2 +/+ control group. The level of CYP2E1 in liver tissue showed a similar pattern to those of the oxidative DNA damage markers. This study shows that acute ethanol consumption increases oxidative DNA damage and that expression of CYP2E1 protein may play a pivotal role in the induction of oxidative DNA damage. The finding that oxidative DNA damage was more intense in Aldh2 –/– mice than in Aldh2 +/+ mice suggests that ALDH2‐deficient individuals may be more susceptible than wild‐type ALDH2 individuals to ethanol‐mediated liver disease, including cancer.

The Effects of 1-Nitropyrene on Oxidative DNA Damage and Expression of DNA Repair Enzymes

The Effects of 1‐Nitropyrene on Oxidative DNA Damage and Expression of DNA Repair Enzymes: Yong‐Dae Kim, et al. Department of Preventive Medicine & Medical Research Center, College of Medicine, Chungbuk National University, South Korea—Nitropyrenes (NPs) present in diesel and gasoline emissions are mutagenic and carcinogenic in experimental animals. Nitro‐reduction of 1‐NP causes oxidative stress. It is unclear whether 8‐hydroxydeoxyguanosine (8‐OH‐dG) is produced from 1‐NP and whether it contributes to the carcinogenic activity of 1‐NP. In this study, we measured the level of reactive oxygen species (ROS) in cultured human lung epithelial cells after exposure to 1‐NP and the intracellular level of 8‐OH‐dG and expression level of the 8‐OH‐dG repair enzymes. As results, 1‐NP induced the generation of 8‐OH‐dG via ROS, but 8‐OH‐dG repair enzymes prevented an increase of 8‐OH‐dG formation in cellular DNA of the A549 cell line below 250 µM of 1‐NP. These data suggest that 1‐NP can induce oxidative DNA damage by generation of ROS, which may play a role in the carcinogenesis induced by 1‐NP. These data also suggest that individuals with impaired DNA repair enzymes might be more susceptible to lung cancer induced by 1‐NP.

Oxidative Stress, hogg1 Expression and NF-κB Activity in Cells Exposed to Low Level Chromium

Oxidative Stress, hogg1 Expression and NF‐κB Activity in Cells Exposed to Low Level Chromium: Yong‐Dae Kim, et al. Department of Preventive Medicine, College of Medicine, Chungbuk National University, South Korea— Chromium compounds are carcinogenic to the human lung, although the detailed biochemical mechanism is still unclear. To understand the carcinogenic mechanism in cells exposed to low level hexavalent chromium, we measured the generation of reactive oxygen species (ROS) and 8‐hydroxydeoxyguanosine (8‐OH‐dG), the transcription of hogg1, which encodes an 8‐OH‐dG repair protein, and NF‐κB activation levels in the A549 human lung epithelial cell line after exposure to Cr (VI) at concentrations of 12.5 to 800 µM. In A549 cells, ROS levels and DNA binding by NF‐κB increased in proportion to the concentration of Cr (VI). These increases were diminished by pretreatment with catalase, superoxide dismutase, or D‐mannitol, but the levels of 8‐OH‐dG and expression of hogg1 did not change significantly with Cr (VI) exposure. These results suggest that the induction of ROS and the activation of NF‐κB are important in the carcinogenic mechanism of Cr (VI), but it is unlikely that Cr (VI) concentrations below 800 µM increase 8‐OH‐dG levels or the expression of hogg1.

Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue

AIM To evaluate the relationship among Helicobacter pylori (H. pylori) infection, CagA status, and dietary factors with RUNX3 promoter hypermethylation. METHODS Gastric cancer tissue samples were collected from 184 South Korean patients. All patients were interviewed following a semi-quantitative food frequency questionnaire. The average frequencies of intake and portion sizes of 89 common food items were documented, and total intakes of calories, nutrients, vitamins, and minerals were calculated for each subject. DNA was extracted from gastric cancer tissue samples, and amplification of the HSP60 gene was performed to detect H. pylori infection. Nested polymerase chain reaction (PCR) was used to detect the presence of the CagA gene. RUNX3 gene expression was measured by reverse transcription-PCR, and RUNX3 methylation status was evaluated by methylation-specific PCR. The odds ratios (ORs) and 95%CI associated with RUNX3 promoter hypermethylation status were estimated for each of the food groups, lifestyle factors, and the interaction between dietary and lifestyle factors with CagA status of H. pylori infection. RESULTS Overall, 164 patients (89.1%) were positive for H. pylori DNA, with the CagA gene detected in 59 (36%) of these H. pylori-positive samples. In all, 106 (57.6%) patients with gastric cancer demonstrated CpG island hypermethylation at the RUNX3 promoter. RUNX3 expression was undetectable in 52 (43.7%) of the 119 gastric cancer tissues sampled. A high consumption of eggs may increase the risk of RUNX3 methylation in gastric cancer patients, having a mean OR of 2.15 (range, 1.14-4.08). A significantly increased OR of 4.28 (range, 1.19-15.49) was observed with a high consumption of nuts in patients with CagA-positive H. pylori infection. High intakes of carbohydrate, vitamin B1, and vitamin E may decrease the risk of RUNX3 methylation in gastric cancer tissue, particularly in CagA- or H. pylori-negative infection, with OR of 0.41 (0.19-0.90), 0.42 (0.20-0.89), and 0.29 (0.13-0.62), respectively. A high consumption of fruits may protect against RUNX3 methylation. CONCLUSION These results suggest that the CagA status of H. pylori infection may be a modifier of dietary effects on RUNX3 methylation in gastric cancer tissue.