尚有許多國家如美國、中國大陸、日本、德國、阿根廷、墨西哥、印度、孟加
拉、智利等均有相同之砷暴露問題。希望政府能大力支持無機砷致病機制的研
究，並鼓勵整合大型研究，收集大樣本並將分子生物醫學之技術應用在台灣地
區研究，以便能傳承過去癌症流行病學研究之光榮歷史領先其他國家。並以臺
灣經驗分享於世界其他國家，進而對世界各地日益增多地下水砷污染及砷誘發
之健康危害事件有實質的幫助。
“The Relationship among Plasma Micronutrients, Antioxidant Enzyme Genetic Polymorphism, Arsenic Methylation Capability and Urothelial
Carcinoma”
Y Y.M. Hsueh1, Y.T. Chen2, H.J. Tsai1, C.J. Chung3, Y.K. Huang2, Y.S. Pu4, C.J. Chen5.
1Department of Public Health, School of Medicine, Taipei Medical University, Taipei, Taiwan,
2Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, 3School of Public Health, Taipei Medical University, Taipei, Taiwan, 4Department of Internal Medicine, National Taiwan University, Taipei, Taiwan, and College of Medicine and 5Graduate Institute of Epeidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan.
Abstract
This study is to examine the potential role and interaction among the antioxidant enzyme polymorphism, plasma micronutrient, arsenic methylation capability, cigarette smoking, oxidative damage 8-OHdG levels and urothelial carcinoma (UC). There were 152 patients with
pathologically proven UC were recruited from the Department of Urology, National Taiwan University Hospital (NTUH) between September 2002 and May 2005. Age and gender matched 158 study subjects without UC were collected from adult health examination at Taipei Municipal WanFang Hospital. Well-trained interviewers did the standardized personal interviews for study subjects who gave their consent based on structural questionnaire and recruited their blood and urine samples. Obtained information including the demographic characteristics, cigarette smoking and alcohol drinking habits, occupational exposure history, and personal and family disease history. DNA was extracted from buffy coat to analyze the manganese superoxide dismutase (MnSOD) and catalase polymorphism utilizing polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP). The urinary concentration of 8-OHdG was assayed by enzyme-linked immunosorbent assays (ELISA). Urine samples were examined by high-performance liquid chromatography (HPLC) to separate arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), and dimethyarsinic acid (DMA) and then quantified by hydride generator combined with atomic absorption spectrometry. Plasma samples were examined by HPLC to analyze micronutrients (retinol, α-tocopherol, β-carotene and lycopene). We found that arsenic methylation capability in case group was significantly worse than control group. We also found the α-tocopherol concentration was inversely related to UC risk. The lower α-tocopherol and the lower secondary methylation capability was the high UC risk. Cigarette smoker with
CI 4.78-58.21). The relationship among MnSOD and catalase genetic polymorphisms and 8-OHdG and UC will be presented in the conference.
Introduction
In Taiwan, urothelial carcinoma (UC) was ranked as the 7th and 10th most common cancer for males and for both sexes, respectively in 2000. The incidence rates of UC have been
progressively increased in the past decades in Taiwan; with the age-specific rates for males and females in 2000 were 10.2 and 4.4 per 105, respectively. It was found a clear dose-response relationship between arsenic water levels and UC mortality and incidence in an arseniasis
endemic area in Taiwan. The capability to metabolize inorganic arsenic differs among individuals.
Whether individual capability of arsenic methylation affects risks of UC among subjects exposed to a previously accepted safe level (50 µg/L) of arsenic is an important issue. Endogenous
defense against ROS includes catalase, and superoxide dismutase (SOD). Micronutrients are antioxidant against ROS. To examine the potential role and interaction among the antioxidant enzymes genetic polymorphisms, plasma micronutrients, arsenic methylation capability, cigarette smoking, oxidative damage 8-OHdG levels and urothelial carcinoma (UC).
Material and Methods
Study Subjects and questionnaire interview
One hundred and forty-three patients with pathologically proven UC (age 24 to 93 years) were recruited from the Department of Urology, National Taiwan University Hospital (NTUH) between September 2002 and May 2004. Age and gender matched 143 control subjects without UC were collected from a hospital-based pool including those with benign diseases (benign prostatic hyperplasia, urolithiasis, urinary tract infection, voiding dysfunctions, etc) from the Department of Urology, NTUH, those receiving senior citizen health examination at Taipei Medical University Hospital and those receiving adult health examination at Taipei Municipal WanFang Hospital.
Well-trained personnel carried out the standardized personal interview that was based on a structured questionnaire. Information obtained included demographic and socioeconomic characteristics, consumption of alcohol, cigarette smoking, occupational and environmental exposure to possible carcinogens, chronic medication history, and personal and familial history of urological diseases. Study subjects who gave their informed consent were subjected to
Frozen urine samples were thawed at room temperature, dispersed by ultrasonic wave, filtered through Sep-Pak C18 column. An aliquot of 200 µL urine was used for separation of arsenic species by high performance liquid chromatography and was quantified by hydride generator-atomic absorption spectrometer (HG-AAS). Arsenic methylation capability was assessed by the percentages of various arsenic species, the primary methylation index (PMI) defined as the ratio between MMA and inorganic arsenic (AsIII + AsV) levels, and the secondary methylation index (SMI) defined as the ratio between DMA and MMA.
Determination of serum antioxidant micronutrient level
Levels of α-carotene, β-carotene, lycopene, α-tocopherol and retinol of serum samples will be measured by high performance liquid chromatography (HPLC) according to the procedure described previously (63). Analysis will be carried out by using reversed-phase HPLC (Hitachi), mobile phase will be methanol: acetonitrite: chloroform = 47: 48 :5, and multiwavelength monitoring. Retinol will be detected at 325 nm, α-tocopherol at 280 nm, and lycopene, α- and β-carotene at 466 nm. Serum samples for each case-control set will be thawed from -70℃
refrigerator in the dim light at room temperature and assayed on the same day to ensure that temporal variability in the laboratory assays would equally affect cases and controls. All laboratory personnel will be unaware of disease status of subjects whose serum samples were tested. In order to obtain reliable analytical result, strict quality control procedures, such as blank analysis, duplicate test, spiked analysis will be carried out.
MnSOD gene polymorphism assay
Genotyping of MnSOD polymophism (a T to C substitution in the mitochondria targeting sequence) was performed by PCR amplification procedure using the primer set of
5’-GCACCAGCAGGCAGCTGGCGCCGG-3’ and 5’-TGCGCGTTGATGTGAGGTTCCAG-3’.
The amplified products were digested with TurboTM NaeⅠ(Progema) and analyzed by
electrophoresis on a 10% polyacrylamide gel. The wild-type allele had no NaeⅠ site and was characterized by a 112 base pair(bp) fragment on gel. 90 and 22 bp fragments on gel
characterized the mutant allele. The heterozygous genotype had both alleles and was characterized by 112, 90 and 22 bp fragments.
Genotyping of catalase polymorphism (a C-to-T substitution of the C262T polymorphic site located on chromosome 11 p 13) was performed by PCR amplification following digestion with Sma I, and analyzed by 4% agarose gel electrophoresis as previously described (Forsberg et al., 2001). Two fragments of 155 and 30 bp were characterized as the wild-type allele and a 185-bp fragment as the mutant allele.
Data analyses and statistical methods
Student’s t test was used to compare the differences in urinary arsenic profile between cases and controls. Univariate logistic regression was performed to compare the distributions of gender, age, cigarette smoking history, the frequencies of alleles and genotypes between cases and
controls. Multivariate logistic regression analysis to adjust for age and cigarette smoking history was performed to calculate odds ratio (OR), and 95% confident interval (CI). SAS Version 8.2 was used for all statistical analyses.
Conclusion
Our study is shown that higher total arsenicals, inorganic arsenics and MMA% in cases than those in controls and the α-tocopherol concentration was inversely related to UC risk. After adjusting for other risk factors, study subjects who are cigarette smoker or have poor arsenic methylation capability with lower VitE or lycopene concentration had significantly higher UC risk. We did not find a relationship among MnSOD and catalase gene polymorphisms, 8-OHdG and UC risk.
Table 1. Sociodemographic characteristics, the MnSOD and catalase genotype distribution in UC cases and healthy controls
UC Cases
Table 2. Distribution of urinary arsenic index, 8-OHdG and micronutrients concentrations in UC patients and health controls
No. UC Cases Control P value
Table 3. The interaction among antioxidants enzymes polymorphisms, smoking status and micronutrients concentration for UC risk.
MnSOD genotype Adjusted-ORa (95%CI)
Catalase genotype Adjusted-ORa (95%CI)
≧ 90.21 WM+MM 0.61(0.29-1.29) WM+MM 0.50(0.12-2.11) Ever 1.76(0.89-3.48)
＜ 90.20 WW 0.62(0.35-1.09) WW 0.81(0.50-1.29) Never 0.72(0.39-1.36)
＜ 90.20 WM+MM 0.93(0.44-2.00) WM+MM 0.78(0.19-3.26) Ever 1.75(0.84-3.61)
VitE (µg/dL)
≧ 1112.41 WW 1.00# WW 1.00# Never 1.00#
≧ 1112.41 WM+MM 0.77(0.32-1.86) WM+MM 0.58(0.11-3.03) Ever 1.26(0.55-2.88)
＜ 1112.40 WW 2.28(1.27-4.07) WW 2.56(1.55-4.23) Never 1.95(1.02-3.73)
＜ 1112.40 WM+MM 2.48(1.14-5.41) WM+MM 2.18(0.54-8.75) Ever 4.69(2.25-9.77) Lycopene (µg/dL)
≧ 8.25 WW 1.00 WW 1.00 Never 1.00#
≧ 8.25 WM+MM 0.81(0.36-1.82) WM+MM 0.21(0.02-1.78) Ever 2.73(1.24-6.00)
＜ 8.24 WW 1.14(0.65-1.99) WW 1.29(0.80-2.09) Never 1.82(0.97-3.44)
＜ 8.24 WM+MM 1.18(0.55-2.52) WM+MM 1.61(0.42-6.07) Ever 2.71(1.35-5.43)
β-carotene (µg/dL)
≧ 17.23 WW 1.00 WW 1.00 Never 1.00
≧ 17.23 WM+MM 0.63(0.29-1.38) WM+MM 0.80(0.23-2.78) Ever 2.26(1.06-4.81)
＜ 17.22 WW 0.69(0.39-1.20) WW 0.93(0.57-1.50) Never 0.99(0.52-1.88)
＜ 17.22 WM+MM 0.92(0.43-1.96) WM+MM 0.46(0.08-2.66) Ever 1.85(0.93-3.67)
a After adjustment for age, sex, smoking status, alcohol drinking and education.; b After adjustment for age, sex, alcohol drinking and education.; # Trent P value<0.05
Table 4. The interaction among antioxidants enzymes polymorphisms, smoking status and micronutrients concentration for UC risk.
Total arsenicals (µg/g creatinine)
a After adjustment for age, sex, smoking status, alcohol drinking and education.; # Trent P value<0.05