中文摘要
前言：多氯聯苯、多氯夫喃、戴奧辛類化學物是廣佈全世界的持久性環境污染物，
影響人類之健康效應甚為嚴重。暴露這些化學物質與所產生的健康效應之間的問 題是我們所關心的重點。在動物實驗中，與這些不良健康效應相關的顯著生物指 標是細胞色素 P450 1 家族。因此此研究主要就是想了解 CYP1 酵素家族成員(如 CYP1A2)的活性強弱是否為暴露多氯聯苯或多氯夫喃後，造成人體健康效應的生 物指標，且此活性的不同是否因個人易感性不同而有所差異。
方法： 在台灣中部，暴露於多氯聯苯及多氯夫喃的油症者，主要是食入有毒的
米糠油所致。因此本研究團隊持續對這群油症中毒者及其對照組進行追蹤個案的 健康情形，並測量個案血液中的多氯聯苯及多氯夫喃濃度。在基因部份，除利用 咖啡因呼吸測試(caffeine breast test)來監測 CYP1A2 的活性；同時也利用聚合酵 素鏈鎖反應(PCR)及限制性片段長度多型性(RFLP)進行 CYP1A1 基因，(分別以 MspI 及 NcoI 兩種限制酶來切)；AhR 基因(以 MseI 限制酶來切)；GST-M1 及 GST-T1 基因的多型性(用 albumin 做為控制)。
結果：由咖啡因呼吸測試可知，暴露組較控制組，提升了 250%的 CYP1A2 活性。
然而，經由迴歸分析可知，CYP1A2 的活性與此化學物質的毒性當量(TEQ)的相 關高達 0.6。同時 CYP1A2 的活性愈高，個案中罹患皮膚問題的比例也愈高，特 別是產生氯痤瘡及不正常的指甲。在四種基因中，只有發現在同型突變的 AhR MseI 基因型的 CYP1A2 活性較不被誘發。
結論：在多氯聯苯或多氯夫喃中，CYP1A2 的活性是一個具代表性的生物指標，
且它的活性會被 AhR 的基因型所影響。
關鍵詞: 多氯聯苯，多氯夫喃，細胞色素 1A2，Ah receptor，單核甘酸多樣性
Abstract
Introduction: PCBs, dioxins, and PCDFs continue to be a world wide public health concern due to their levels in the environment and humans, and associated adverse health effects. A major concern has been the identification of the degree of potential adverse health effects from an exposure. In animal models, one of the most sensitive biomarkers of physiologically significant body burdens and adverse health effects has been the capacity of these chemicals to induce cytochrome P450 1 family of enzymes.
This study examined whether the capacity of P450 family 1 enzymes (e.g. CYP1A2) to be biomarkers of exposure to a mixture of PCBs and PCDFs and of adverse human health effects, and whether such induction of capacity dependent on genotypes.
Method: Yucheng (‘oil-disease’) victims were Taiwan central people exposed to polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs), from the ingestion of contaminated rice oil in 1978–1979. We followed up these exposure and control subjects and compared to congener specific blood levels and the subjects’
medical history. The caffeine breath test (CBT), a sensitive monitor of cytochrome P451A2 (CYP1A2) activity was conducted. We use PCR (Polymerase chain reaction) and RFLP (Restriction fragment length polymorphism) for AhR (MseI) and CYP1A1 (MspI, NcoI); multiplex PCR for GSTM1 and GSTT1(null/non-null), using albumin as positive control to test single nucleotide polymorphism of four genes.
Results: The results showed that CYP1A2 activity measureed by CBT was elevated in Yucheng subjects by 250%, and correlated with serum TEQ (R2= 0.6) by regression model. Manifestations like chloracne and nail abnormality were well predicted by TEQ and P4501A2 activity. People with homozygous mutant genotype in AhR MseI site were less inducible in CYP1A2 than other genotypes.
Conclusion: We conclude that induced CYP1A2 can be a phenotypic biomarker of PCBs/PCDFs exposure, and such induction is affected by AhR genotype.
Key word: polychlorinated biphenyls (PCBs) ， polychlorinated dibenzofurans (PCDFs)，CYP1A2，AhR，single nucleotide polymorphism (SNP)
目錄 前言
研究目的 文獻探討 研究方法 結果
結論及討論 計畫成果自評 附錄一:圖、表 附錄二：發表之文章 參考文獻
前言
Polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs) and related compounds are among the most widespread environmental pollutants (Jeason, 1987); they have been routinely detected in wildlife as well as human tissue samples since the 1960s. Multiple adverse health outcomes including those of the skin, liver, neurological, and reproductive systems have been reported in humans and animals.
Due to their long half lives in the environment and in animals and humans, these environmental chemicals will be a concern for many years to come. The toxic capacity of these chemicals to the human have been a world wide medical, public health, and legal concern resulting in hundreds of millions of dollars in the clean up of these potentially harmful chemicals.
研究目的
Our studies examined
1. Whether the capacity of P450 family 1 enzymes (e.g. CYP1A2) to be biomarkers of exposure to a mixture of PCBs and PCDFs and of adverse human health effects, and whether such induction of capacity dependent on genotypes.
2. Whether AhR, CYP1A1 and GSTM1/T1 genotypes predict health outcomes e.g.
skin manifestations.
文獻探討
In the animal models, some of the health effects have been shown to correlate with the capacity of the congener mix to induce the cytochrome P450 family one enzymes through the interacting with the Ah receptor. The degree of P450 family one induction by these chemicals in the human has not been published in most cohorts with known exposure to high levels of PCBs, PCDFs, PBBs, or dioxins.
In 1978-9, a mass poisoning occurred in central Taiwan from rice oil contaminated by heat-degraded PCBs (Hsu, 1985). Symptoms included chloracne, hyperpigmentation, and peripheral neuropathy, and the illness was referred to as "Yucheng" (oil disease in Chinese). By 1983, a registry set up and maintained by the Taiwan Provincial Department of Health included 2061 subjects (Hsu, 1994). Patients used the contaminated cooking oil for an average of 8 months; they were estimated to have consumed about 1 gram of PCBs and 3.8 mg of PCDFs (Lan, 1981) (mostly the 2,3,4,7,8-penta-CDF and the 1,2,3,4,7,8-hexa-CDF) during that time, resulting in median serum PCB levels of 40 to 60 parts per billion wet weight, and serum PCDF levels of about 2.7 parts per trillion (ppt) wet weight for the penta-CDF and 10.8 ppt wet weight for the hexa-CDF. Some of these chemicals are extremely persistent in
human tissue; 14 years after exposure, PCB and PCDF measurement in 56 women from the cohort showed that total PCBs were still 7 fold higher and PCDF congeners 40-130 times higher than that of pooled local controls (Guo, 1997). Persons
poisoned in this incident had excess morbidity of several conditions. Most prominent were the skin manifestations including chloracne, abnormal nails, and hyperkeratosis (Guo, 1999).
Genetic polymorphisms might play some roles in human susceptibility to dioxin-like chemicals. Aryl hydrocarbon receptor (AhR) has been known as a ligand-activated receptor for TCDD and dioxin-like chemicals, such as coplanar PCB congeners and PCDFs (Reyes, 1992). The toxic potency of dioxin and PCB congeners is highly related to binding affinity to AhR. AhR plays an important role to activate the transcription of xenobiotic metabolization as well as other genes (e.g. Cytochrome p450 family).
研究方法
We used PCR (Polymerase chain reaction) and RFLP (Restriction fragment length polymorphism) for AhR and CYP1A1; multiplex PCR for GSTM1 and GSTT1, using albumin as positive control to determination of genetic polymorphisms. CYP1A2 activity by caffeine breath test (CBT): Non-radioactive [3-13C-methyl] caffeine (99%
13C) was given to the subjects at a dose of 3 mg/kg. A breath sample was obtained by having the subjects blow into a plastic bag just prior to and after the ingestion of the labeled caffeine at 30 and 60 min. CYP1A2 activity as indicated by 3-N-demethylation, i.e. 13CO2/12CO2 ratio was determined by differential gas-isotope ratio mass spectrometry. The 1-hr accumulative exhalation of labeled CO2 was the CBT parameter calculated. Serum levels of PCB, PCDF, and PCDD congeners: The stored serum samples, drawn in 1994, were sent on dry ice to the U.S.
Centers for Disease Control and Prevention, for measurements of dioxin-like chemicals by high-resolution gas chromatography/mass spectrometry. TCDD toxic equivalency (TEQ) was calculated according to the WHO-TEQ system. For controls, males were estimated as 14 ng/kg lipid for PCDFs, and females as 16 according to previous studies.
結果
Demographic characteristics- for genotypic predisposition study
A completely telephone interview for 795 Yucheng subjects and 693 matched control subjects were obtained, and the lifetime prevalence of chloracne, abnormal nails, hyperkeratosis, skin allergy, liver diseases and other symptoms in 1993 were recorded.
Among them, 574 subjects (393 Yucheng and 181 controls) had genetic
polymorphisms of five genes determined. Table 1 compared the demographic and personal characteristics of Yucheng and control groups. Stratified with sex, there is no significant difference in age, education level, smoking and drinking habits in both groups. The median of PCB exposure levels in 1980-1982 (51 ppb) was far higher than that in general population (1.67 ppb) documented in 1997.
Demographic characteristics- for 1A2 phenotypic study
Caffeine breath test was done in 308 subjects (174 exposed and 134 controls) in 1995 Age, education, and smoking history in Yucheng and control subjects at time of caffeine breath test are shown in table 2.
Skin Manifestations related to exposure level
Among all subjects, 518 could be classified by PCB level in 1980-1982 into three groups, included 181 control group; 169 with intermediate exposure (mean of PCB level: 29.0 ± 7.3 ppb); 168 highly exposed (mean of PCB level: 140.0 ± 7.4 ppb). The review of systems showed that chloracne, abnormal nail, hyperkeratosis and skin allergy were significantly increased in Yucheng groups when hyperkeratosis, abnormal nail, and chloracne were rather rare in control population. These skin problems were dose-related to PCB exposure level (Figure 1).
Relationship between CYP1A2 activity, exposure levels, and skin manifestations Caffeine breath test was done in 308 subjects (174 exposed and 134 controls) in 1995.
The CBT parameter used for comparisons was % C13-labeled exhaled over one hour and was compared by gender groups, smoking status, and age in exposed and unexposed subjects (Table 3). Yucheng subjects had higher CBT parameter than the controls by 250%. This is not changed by stratifying subjects by sex or smoking status. In unexposed people, smokers had higher rate of CYP1A2 activity.
Regression analysis was done between CBT parameter (% C13-labeled exhaled over one hour) and serum levels of TCDD TEQ. CBT parameter is highly associated with serum TEQ (Figure 2).
The relationship between reported clinical manifestations and CBT parameters were compared by logistic regression. Among Yucheng subjects, the CBT parameter was categorized into <3% (N=103), 3-4% (N=41), and >4% (N=33). Risks of having abnormal nails and chloracne were associated with increased CBT parameters of greater than 4, with OR of 3.90 and 3.09 respectively. Borderline significance was found between gum pigmentation and CBT parameter of greater than 4 (Table 4).
Genetic predisposition to CYP1A2 induction
Among those genotypes we tested, AhR MseI homozygous mutant was associated with lower CYP1A2 inducibility than wild/wild and wild/mutant genotypes (Figure 3).
結論及討論
Since exposure to dioxin-like chemicals is associated with induction in metabolizing enzymes CYP1A2, as measured by caffeine metabolism, and such induction is associated with clinical findings of PCB/PCDF intoxication, CBT seems to be an excellent biomarker of exposure to PCBs and PCDFs. Induction of CBT seemed to be different between different AhR genotypes, suggesting different susceptibility among these subjects.
計畫成果自評
本計畫執行三年期間，從第一年的個案資料收集、基因型分析；第二年的咖啡因 呼吸測試及資料統整；到最後一年的論文發表，共發表了六篇文章，雖文章內容 方向與原設定目標不盡相同，但經研究團隊不斷地修正，使得結果更趨近事實。
雖不盡完美，但也相當符合我們計畫的預期目標。
.
參考文獻
1. Jensen AA. Polychlorinated biphenyls (PCBs), polychlorodibenzo-p-dioxins (PCDDs) and polychlorodibenzofurans (PCDFs) in human milk, blood, and adipose tissue. Sci Total Environ 64: 259-293 (1987).
2. Hsu ST, Ma CI, Hsu SKH, Wu SS, Hsu NHM, Yeh CC, Wu SB. Discovery and epidemiology of PCB poisoning in Taiwan: A four-year follow up. Environ Health Perspect 59:5-10 (1985).
3. Hsu CC, Yu ML, Chen YC, Guo YL, Rogan WJ. The Yu-cheng Rice Oil
Poisoning Incident. In: Schecter A, ed. Dioxin, Related Chemicals, and Health.
New York:Plenum, 661-684. (1994).
4. Lan CF, Chen PH, Shieh LL, Chen YH. An epidemiological study on polychlorinated biphenyls poisoning in Taichung area. Clin Med 7:96-100 (1981).
5. Guo YL, Ryan JJ, Lau BPY, Yu ML, Hsu CC. Serum levels of PCB/PCDF congeners 14 years after accidental exposure to contaminated rice oil. Arch Environ Contamin Toxicol 33:104-108 (1997).
6. Guo YL, Yu ML, Hsu CC, Rogan WJ. Goiter, skin diseases, arthralgia, and anemia after PCB/PCDF poisoning: 14 year Follow-up of the Taiwan Yucheng cohort. Environ Health Persp 107:715-719 (1999).
7. Reyes H, Reisz-Porszasz S, Hankinson O. Identification of the Ah receptor nuclear translocator protein (ARNT) as a component of the DNA binding form of the Ah receptor. Science 256:1193-1195 (1992).
附錄一:圖、表
Table 1. Demographic and personal characteristics of Yucheng and control subjects of 1993 in Taiwan.
sex Male Female
exposure Yucheng (n=176)
Control (n=100)
p Yucheng (n=217)
Control (n=81)
p
Age (yr) a 50.8 ± 0.9 53.1 ± 1.2 n.s. 45.4 ± 0.8 47.5 ± 1.3 n.s.
30-39 (%) 22 18 48 36
40-49 (%) 21 20 14 21
50-59 (%) 29 28 n.s. 24 25 n.s.
>60 (%) 28 34 14 18
Education (yr) a 6.9 ± 0.3 7.1 ± 0.4 n.s. 5.3 ± 0.3 6.1 ± 0.5 n.s.
Ever smoked b 106 (60.6%) 67 (67.0%) n.s. 2 (1.0%) 4 (4.9%) n.s.
Ever drinking b 93 (52.9%) 50 (50.0%) n.s. 21 (10.7%) 11 (13.6%) n.s.
PCB level (ppb)c 57.4 - 48.0 -
a. age and years of education were compared by unpaired t-test (mean ± standard).
b. age distribution, smoking history and drinking history were compared by chi-square test (values in parentheses are percent).
c. PCB levels were measured by median of level in 1980-2.
Table 2. Age, education, and smoking history of Yucheng and control subjects, at time of caffeine breath test, 1995, Taiwan.
Men Women
Yucheng Reference p-value Yucheng Reference p-value
Subject No. 71 48 103 86
Age, year 53.3 + 12.6 54.3 + 13.1 n.s. 45.3 + 11.9 43.8 + 11.3 n.s.
Distribution
30-39 (%) 19.7 25.0 52.4 58.1
40-49 (%) 22.5 8.3 19.4 17.4
50-59 (%) 21.1 35.4 n.s. 10.7 12.8 n.s.
60-69 (%) 28.2 16.7 14.6 8.1
>70 (%) 8.5 14.6 2.9 3.5
Education, yr 7.3 + 3.6 7.7 + 3.7 n.s. 6.4 + 4.0 6.4 + 4.7 n.s.
Currently smoking
42.2% 54.2% n.s. 1.0% 2.3% n.s.
Serum levels (ppt) (GM + GSD)
Dioxin TEQ 0.5 + 6.9 1.5 + 10.2
Furan TEQ 216.0 + 2.5 316.7 + 4.7
PCB TEQ 5.1 + 2.6 3.3 + 3.0
Total TEQ 229.0 + 2.5 14 361.0 + 3.5 16
Table 3. CYP1A2 activity as determined by cumulative exhalation of labeled C13 in one hour by exposure, gender, and smoking status. Tests were done using Wilcoxon rank-sum test.
Exposed Unexposed exposed vs.
N N unexposed
Gender Male 71 2.46 + 0.14 48 1.30 + 0.17 P<0.001 Female 103 3.20 + 0.12 86 1.12 + 0.13 P<0.001 Currently smoking No 143 2.96 + 0.14 106 1.17 + 0.07 P<0.001 Yes 31 2.88 + 0.30 28 1.46 + 0.12* P<0.001
Age Non-significant Non-significant
Male non-smokers 2.30 + 0.20 1.13 + 0.29 P=0.0011 Male smokers 2.86 + 0.25 1.48 + 0.28 P<0.001 Female non-smokers 3.20 + 0.12 1.12 + 0.13 P<0.001
*p<0.05 compared with non-smokers
There were only 1 female smoker in exposed group and 2 in control, thus comparisons were not done.
Table 4. CBT parameter as a predictor of risk of having clinical manifestations in Yucheng patients.
CBT Abnormal nail Skin allergy Chloracne Hyperkeratosis Gum pigmentation
<3% 1 1 1 1 1
3-4% 2.04 (0.66-6.10) 0.68 (0.26-1.66) 1.43 (0.52-3.76) 2.45 (0.65-8.92) 1.42 (0.27-6.25)
>4% 3.90* (1.37-11.3) 1.11 (0.44-2.68) 3.09* (1.21-7.90) 2.84 (0.75-10.5) 3.63 (0.99-13.9)
0 5 10 15 20 25 30
chloracne abnor
mal nail
hyperkeratosis
skin allergy PCB level(ppb)
disease(%)
control intermediate high
Figure 1. Disease prevalence according to PCB levels in 1980-82.
*p-value <0.001 by Chi-square test in three groups.
PCB exposed group were divided into “intermediate”: PCB level <51 ppb and “high”:
PCB level >=51 ppb
*
*
*
*
0 1 2 3 4 5 6
% labeled C13 exhaled in 1 hr
Figure 2. CBT parameter (% labeled C13 exhaled in one hour) by serum level of TCDD toxic equivalency (ng/kg TEQ). Regression equation: CBT parameter = 1.23 + 0.0032 * TEQ. R2 = 0.60. P<0.001.
Figure 3. CBT parameter (% labeled C13 exhaled in one hour) by serum level of TCDD toxic equivalency (ng/g TEQ). Green dot: AhR MseI W/W or W/M genotypes, red dot: AhR MseI MM genotype, P<0.05 comparing the regression coefficient of 2 lines
附錄二：發表之文章
1. Tsai PC, Huang WY, Lee YC, Guo YL. Genetic polymorphisms in CYP1A1 and GSTM1 predispose humans to PCBs/PCDFs-induced skin lesions.
2. Lambert GH, Needham LL, Turner W, Lai TJ, Guo YL. Induced CYP1A2 activity as a phenotypic biomarker in humans exposed to PCBs/PCDFs.
3. Guo YL, Lambert GH, Hsu CC, Hsu MM. Yucheng: health effects of prenatal exposure to polychlorinated biphenyls and dibenzofurans. Int Arch Occup Environ Health 77: 153–158 (2004).
4. Guo YL, Hsu PC, Huang WY, Lee YC, Wang SH. Kuo PL. Polychlorinated biphenyls and dibenzofurans exposure does not induce sperm aneuploid in humans.
5.Hsu PC, Lai TJ, Guo NW, Lambert GH, Guo YL. Serum Hormones in Boys Prenatal Exposed to Polychlorinated Biphenyls and Dibenzofurans.
6. Hsu PC, Lai TJ, Guo NW, Lambert GH, Guo YL. Serum hormones in boys prenatally exposed to polychlorinated biphenyls and dibenzofurans. J Toxicol Environ Health A 2005, in press.
0 1 2 3 4 5 6
95.CBT
0 .1 .2 .3 .4 .5 .6 .7 .8 .9
Sqrt 95CDC DF TEQ
