氯乙烯聚合工人之易感性標記研究 (二)
Study on the Susceptibility Biomarkers in Polyvinyl Chloride Workers 計畫編號：NSC88-2314-B-002-121 執行期限：自民國 87 年 8 月 1 日起至民國 88 年 7 月 31 日止 主持人：鄭尊仁 執行機構及單位名稱：國立台灣大學公共衛生學院職業醫學與工業衛生研究所 中文摘要 氯乙烯單體暴露在聚氯乙烯工人 引起肝功能異常並不一致，可能與代 謝基因,微量元素，B 型、C 型肝炎病 毒交互作用有關。動物實驗指出氯乙 烯可能被 CYP2E1、ALDH、EH 和 GSTs 酵素所代謝。先前我們已經證實了氯 乙烯工人的 CYP2E1、GSTT1 和 ALDH2 基因型與其肝功能異常有關，但是與 血清抗氧化劑微量元素無顯著相關。 在這個研究中，我們收集了更多的樣 本來進一步探討代謝基因型包括新發 現 EH（EH exon 3，4)基因型、微量營 養素及 B、C 型肝炎病毒與氯乙烯聚合 工人肝功能異常之相關，在五家氯乙 烯製造工廠中，共收集了 287 名男性 工人，並依其職務名稱與空氣濃度區 分為高暴露組（TWA＞1 ppm）和低暴 露組（TWA＜1 ppm）。藉由問卷收集 其抽煙、飲酒、職業史與疾病史資料， 並 且 以 血 清 中 ALT （ Alanine aminotransferase）濃度來當作肝功能指 標；此外，B 型 及 C 型肝炎病毒感染 標記也一併分析。CYP2E1，ALDH2， EH (Exon 3 及 4) 和 GSTT1 基因型是 以 PCR/RFLP 來測定。微量營養素則 包括維生素 A、維生素 E、α-胡蘿蔔 素和β-胡蘿蔔素，其血清濃度係以 HPLC 來測定。結果顯示 CYP2E1， GSTT1 和 ALDH2 與氯乙烯聚合工人 的 ALT 異常有關而 EH 基因型則與肝功 能無關。同時抗氧化劑與氯乙烯工人 肝功能異常也無相關，但是進一步分 析發現在 B 型肝炎 C 型肝炎感染工人 中，血中去氧化劑濃度較低，同時兩 者之間對 ALT 之異常具交互作用。本 研究顯示氯乙烯工人之肝功能受到代 謝基因 ， 病 毒感 染 與 微 量 之 交 互 作 用，具肝炎病毒感染的工人可能須加 強抗氧化劑之攝取。不過仍須進一步 研究來證實。 關 鍵 字 ： 氯 乙 烯 、 CYP2E1 、 GSTT1、ALDH2、EH 基因型、微量元 素 Abstr act Abnormal LFT varies in VCM exposed workers. The role of inherited metabolic traits, micronutrients and hepatitis B and C virus infection on liver damage in VCM workers has not been thoroughly investigated. In the previous study, we found CYP2E1, GSTT1 and ALDH2 were associated with abnormal ALT. However, antioxidants were not associated with abnormal ALT. Here we included newly found Epoxide hydrolase (EH) to investigate if genotypes, micronutrient and hepatitis virus infection were associated with liver damage. In this study, 287 male
workers from five PVC factories were classified into high (time weight average, TWA>1 ppm), and low VCM exposure (TWA<1 ppm). Questionnaires were administered to obtain detailed information. The activity of serum alanine aminotransferase (ALT) was used as a marker of liver damage. Markers of hepatitis B and C virus infection were also analyzed. CYP2E1, ALDH2, EH and GSTT1 genotypes were identified using the polymerase chain reaction. Vitamin A, vitamin E, α-carotene, and β-carotene were measured by HPLC. The results showed that CYP2E1 c2c2, GSTT1 null, ALDH2 1-2/2-2 genotypes were associated with increased ALT abnormality whereas genotype EH was not associated with abnormal ALT. Again, antioxidants were not associated with VCM-induced liver damage. However, hepatitis virus infection had a significant interaction with antioxidant on abnormal ALT.
Key Words: vinyl chloride monomer, CYP2E1, GSTT1, ALDH2, EH, antioxidants, hepatitis B and C infection
Introduction
Studies have shown that vinyl chloride monomer (VCM) exposure can cause liver damage (1,2). Alanine amino-transferase (ALT) is one of the most sensitive and specific biomakers for liver damage.
In the liver, VCM may be metabolized by CYP2E1, ALDH2, GSTs, and EH (3,4,5). These metabolic
enzymes have been reported to be associated with carcinogenesis induced by various chemicals (6). However, the role of inherited metabolic traits on liver damage in VCM workers has not been thoroughly investigated. Our study have showed that CYP2E1, ALDH2 and GSTT1 may be associated with abnormal ALT. Recently, EH was found to be associated with hepatocellular cancer and emphysema (7,8). EH is responsible for the hydroxylation of epoxide leading to detoxification, although sometimes this kind of metabolism can lead to higher toxicity. Here, we included EH genotypes to investigate if this genotypes is associated with VCM induced-abnormal ALT.
Additionally, there is compelling evidence that diets rich in fresh fruits and vegetables may lower the risk of cancers and coronary heart diseases (9). Antioxidants including vitamin A, vitamin E and carotene may be res-ponsible for this effect, although the previous study did not show an association between antioxidant and ALT. In this study, the interaction between micronutrients and hepatitis virus infection on the elevation of liver enzymes was further investigated.
Mater ials and Methods
Study Subjects: A total of 287 male workers, with greater than six months of VCM exposure from five plants, were included for analysis. Detailed
information was collected from interviewer administered questionnaires, after informed consent was obtained. Alcohol drinking was defined as alcoholic consumption at least one time and more than 80 grams of alcohol weekly. In this study, subjects were divided into high (TWA>1 ppm) and low VCM (TWA<1 ppm) exposure groups.
Liver F unction Tests: Alanine amino-transferase (ALT) was used as a marker of liver damage, while HBsAg and anti-HCV were also assayed.
Metabolic Genetic Polymorphism Genetic polymorphisms for CYP2E1, ALDH2 and EH (Exon 3 and exon 4) were determined by PCR/RFLP. GSTT1 genotypes were determined by the presence of these genes after PCR.
Deter mination of plasma micro-nutr ients levels: Micromicro-nutrient levels including vitamin A, vitamin E, α-carotene, β-carotene were measured by high-performance liquid chromatogra-phy.
Statistical Analysis
Odds ratios (OR) and their 95% confidence intervals (C.I.) were computed to examine the association of liver damage with various variables. Logistic regression was used to estimate ORs after adjusting covariates.
Results
287 male subjects were included in the analysis. Individuals of high VCM exposure had higher ALT level than those with low exposure. CYP2E1 c2c2 genotypes in high VCM exposure had the highest proportion of abnormal ALT than other combination of CYP2E1 genotype with VCM exposure (OR=3.5, 95% C.I.=2.5~19.8). While ALDH2, GSTT1 and EH alone were not associated with abnormal ALT. When individuals with both CYP2E1 c1c1/c1c2 genotypes and ALDH2 1-1 genotype were used as reference, OR was 8.3 (95% C.I.=1.9~42.9) for those with both CYP2E1 c2c2 genotypes and ALDH2 1-2/2-2 genotypes; OR was 1.9 (95% C.I.=0.5~ 7.9) for those with both CYP2E1 c2c2 genotype and ALDH2 1-1 genotype, and 1.4 (95% C.I.=0.9~3.7) for those with both ALDH2 1-2/2-2 genotypes and CYP2E1 c1c1 genotype.
In contrast, vitamin A, vitamin E, α-carotene and β-carotene levels were not associated with ALT abnormality. Interestingly, those with hepatitis B and C infection had lower antioxidant level. Further analysis showed that antioxidant level and hepatitis infection had interaction. When stratified by hepatitis infection, in group of negative hepatitis infection, those who had low antioxidant level did not had higher risk of abnormal ALT, However, in group of positive hepatitis infection, those with low vitamin A or vitamin E levels tend to have higher prevalence of abnormal
ALT (p < 0.02, p < 0.04, respectively),
compared with those with high vitamin A or vitamin E levels.
Discussion
In this study, CYP2E1 c2c2 together with GSTT1 null as well as ALDH2 1-1/2-2 were associated with increased abnormality of liver enzymes. Interestingly, susceptible ALDH2 and CYP2E1 had more than additive effect for liver damage. This indicates that VCM is metabolized by CYP2E1 and ALDH2 on the same metabolic pathway. The role of ALDH2 in the health outcomes of VCM workers need further investigation, because one half of Chinese do not have adequate activity for this enzyme (10). Although EH was reported to be responsible for the hydroxylation of epoxide in the previous study, we did not observed an association between EH and abnormal ALT. The major metabolic pathway for VCM may be through the transformation of chloroethlene oxide (CEO) to chloroacetaldehyde (CAA). Thus, EH did not affect ALT.
The association of antioxidant and VCM- related abnormal liver enzyme is not shown in this study. Decreased serum antioxidant in hepatitis virus infection indicate that inflammation caused by virus may generate free radical leading to a decrease in antioxidants. The interaction of hepatitis virus infection with serum antioxidant on abnormal ALT suggest that other than
antioxidants, hepatitis virus infection may also contribute to abnormal ALT through other pathways. Further study may shed light on the mechanism.
Reference
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