行政院國家科學委員會專題研究計畫成果報告

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行政院國家科學委員會專題研究計畫成果報告

無機砷暴露，抗氧化維生素、脂質及宿主易感受性對動脈粥狀硬化危險性交互作用之分子流行病學研究

Arsenic exposure, null genotypes of glutathione S-transferase M1, T1 and P1, and risk of carotid atherosclerosis among residents in Lanyang

Basin of Taiwan

計畫編號：NSC-89-2320-B-038-005, NSC-89-2314-B-038-060, NSC-90-2320-B-038-044 執行期間：88 年 8 月 1 日至 91 年 7 月 31 日

主持人：邱弘毅台北醫學大學公共衛生學系

共同主持人：陳建仁台大公共衛生學院流行病學研究所

計劃參與人員：周怡利、王慧婷、徐憶驊台北醫學大學公共衛生學研究所

中文摘要

本研究的主要目的在探討經由飲用含砷井水與頸動脈粥狀硬化之相關性。

此外，研究中也評估人類易感受基因 GSTM1、T1、P1 及 P53 與砷暴露參予所造成的影響。研究對象共 605 人，其中包含男性 289 人、女性 326 人。在調整年齡、

性別後發現飲水砷濃度 ≧ 100 μ g/L 及 50.1-99.9μg/L 這兩組都有顯著的頸動脈粥狀硬化疾病罹病危險性。此外，調整年齡、性別後研究對象之 GSTP1 為 W/M 及 M/M 基因型者其罹患頸動脈粥狀硬化的 OR 值為 2；P53 為 W/M 及 M/M 基因型者其調整後的 OR 值為 1.9，且均達統計上的顯著差異。GSTP1 之基因型為 W/M 及 M/M 且含砷井水濃度為≧100μg/L 及 50.1-99.9 μg/L 之研究對象的頸動脈粥狀硬化罹病危險性分別為 4.1 及 3.2 且具統計上的顯著差異。P53 基因型為 W/M、M/M 且飲用水含砷井水濃度為≧50μg/L 以上者罹患頸動脈粥狀硬化的罹病危險性為對照組的 3 倍，由此可見環境中砷暴露與人類易感受基因與頸動脈粥狀硬化之間確有明顯的交互作用存在。

關鍵詞：砷、麩氨基硫化合物、粥狀動脈硬化

Abstract

The specific aim of this study is to elucidate the association between arsenic exposure through drink well water and carotid atherosclerosis. In addition, the joint effect between human susceptible genes including GSTM1, T1, P1, and P53 and arsenic exposure is also evaluated. A total of 605 study subjects included 289 men and 326 women were recruited in this study. A significant age-sex-adjusted odds ratios of risk of carotid atherosclerosis were observed both in exposure groups with arsenic concentration in well water greater than 100 and ranged from 50.1 to 99.9 ug/L. In addition, a significant higher age-sex-adjusted odds ratio of 2.0 for the development of carotid atherosclerosis was observed among study subjects with genotypes of W/M and M/M of GSTP1 and 1.9 for study subjects with genotypes of W/M and M/M of P53. Significant age-adjusted odds ratios of 4.1 and 3.2 for the development of carotid atherosclerosis were also found for arsenic concentrations in well water of 50.1-99.9 and >100 ug/L , respectively, among study subjects with W/M and M/M genotypes of GSTP1. A significant three times risk for development carotid

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atherosclerosis was also observed among study subjects who drank well water with arsenic content greater than 50 ug/L and with W/M or M/M genotype of P53 compared with referent group.

key words: Arsenic, glutathione , S-transferase , atherosclerosis

1. Introduction

Arsenic is widely distributed in nature and mainly transported in the environment by water. The main source of arsenic exposure for the general population is through ingestion of water containing high level of inorganic arsenic and of seafood contains high level of organic arsenic. However, organic arsenic is less toxic than inorganic arsenic. (World Health Organization, 1981;

US Public Health Service, 1989). The tentative MCL for arsenic in drinking water set by the US Environmental Protection Agency is 0.05 mg/L. Inorganic arsenic has also been well documented as one of the major risk factors for BFD, a unique peripheral vascular disease identified in the endemic area of arseniasis located on the southwest coast of Taiwan where residents had used high-arsenic artesian well water for more than 50 years (Tseng, 1968; Chen et al., 1988a). The pathological types of BFD include arteriosclerosis obliterans (70％) and thromboangiitis obliterans (30 ％ ) which developed from severe underlying systemic arteriosclerosis (Tseng et al., 1961; Yeh and How, 1963). The atherogenic effects of arsenic has also been well documented.

Ingested inorganic arsenic through drinking water has been related to the development of peripheral vascular disease in Poland, Chile, Mexico, Argentina, Japan and Xinjiang, China (World Health Organization, 1981;

Chen and Lin, 1994; Engel et al., 1994;

Cebrian et al., 1994; Borgono et al., 1977;

Hotta et al., 1989; Wang and Huang, 1994) and among Moselle vintners exposed to inorganic arsenic through contaminated wine (Grobe, 1976). Current studies have also reported long-term exposure to arsenic in drinking water was significantly associated with risk for development ischemic heart disease, cerebrovascular disease and peripheral vascular disease in Taiwan, showing a dose-response relationship( Wu et al., 1989; Chen and Wang, 1990; Tseng et al., 1996; Chen, et al., 1996; Chiou 1997a). In addition, the association between ingestion arsenic through drinking water and hypertension and diabetes mellitus has also been reported in Taiwan and Bangladesh (Lai et al., 1992; Chen et al., 1995; Rahman et al., 1998, 1999). Glutathione (GSH) S-transferases (GST) are a large family of phase II detoxification enzymes that catalyze the conjugation of reduced GSH to a wide spectrum of hydrophobic and electrophilic compounds. There are four subclasses of GST in mammalian cells, namely alpha, mu, pi and theta (Board et al., 1990). Many studies have suggested that GSH might be involved in the initial reduction of arsenate to arsenite and the subsequent oxidative methylation. GSH is necessary enzyme for arsenic methylation, perhaps through the formation of arsenite that is the preferred arsenic form for methylation, or through conjugation with arsenic (Buchet and Lauwerys, 1985, 1987; Thompson, 1993).

Humans with null genotypes of GSTM1, T1, and P1 have been considered to be a high risk group of cancers due to their GSH deficiency.

The specific aim of this study is to evaluate the synergistic effects of arsenic exposure through drinking water and genetic polymorphisms of GST M1, T1, and P1 on the risk of carotid atherosclerosis.

2. Results and Discussion

A total of 605 study subjects included 289 men and 316 women were recruited in

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this study. As shown in Table 1, compared with youngest age group, the significant odds ratios of 5.9 and 2.3 were observed for the oldest and middle age groups, respectively.

Significant risk for occurrence of carotid atherosclerosis was also observed among hypertension patients with age-sex-adjusted odds ratio of 2.4. In addition, men, alcohol drinkers, and patients with diabetes mellitus also had higher but not significant risk for the development of carotid atherosclerosis. Table 2 showed that a significant age-sex-adjusted odds ratios of risk of carotid atherosclerosis were observed both in exposure groups with arsenic concentration in well water greater than 100 and ranged from 50.1 to 99.9 ug/L.

Moreover, study subjects with cumulative arsenic exposure greater than 1.0 mg/L-year also had significant age-sex-adjusted risk of developing carotid atherosclerosis. As illustrated in Table 3, a significant higher age-sex-adjusted odds ratio of 2.0 for the development of carotid atherosclerosis was observed among study subjects with genotypes of W/M and M/M of GSTP1 and 1.9 for study subjects with genotypes of W/M and M/M of P53. Significant age-adjusted odds ratios of 4.1 and 3.2 for the development of carotid atherosclerosis were also found for arsenic concentrations in well water of 50.1-99.9 and >100 ug/L , respectively, among study subjects with W/M and M/M genotypes of GSTP1.. However, study subjects with null genotype of GSTM1 and T1 did not have higher risk of carotid atherosclerosis. Table 4 illustrated that study subjects who drank well water contained arsenic level greater than 50 ug/L and with W/M or M/M genotype of GSTP1 had 2.7 folds risk of developing carotid atherosclerosis compared with those who drank well water contained arsenic concentration equal and less than 50 ug/L as referent group. Significant age-adjusted odds ratios of 4.1 and 3.2 for the development of carotid atherosclerosis were also found for arsenic concentrations in well water of

50.1-99.9 and >100 ug/L , respectively, among study subjects with W/W genotype of GSTP1. A significant three times risk for development carotid atherosclerosis was also observed among study subjects who drank well water with arsenic content greater than 50 ug/L and with W/M or M/M genotype of P53 compared with referent group.

The atherogenic effects of ingestion inorganic arsenic through drinking water have been well documented. A serious studies carried out in Taiwan have reported the significant association between long-term exposure to arsenic in drinking water and the risk for the development of atherosclerotic vascular diseases such as ischemic heart disease, cerebrovascular disease, and peripheral vascular disease, showing a dose-response relationship( Wu et al., Chen, 1990; Tseng et al., 1996; Chen, et al., 1996;

Chiou 1997). GST are a large family of phase II detoxification enzymes that catalyze the conjugation of reduced GSH to a wide spectrum of hydrophobic and electrophilic compounds. They play important roles in protection mechanisms against chemical carcinogenesis. Many studies have suggested that GSH might be involved in the initial reduction of arsenate to arsenite and the subsequent oxidative methylation. In this study, a significant high risk of developing carotid atherosclerosis was observed among study subjects with W/M or M/M genotypes of GSTP1 and P53. Humans with null genotypes of GST P1 and P53 have been considered to be a high risk group of carotid atherosclerosis due to their GSH deficiency.

Self-evaluation of this study

A significant high risk of developing carotid atherosclerosis was observed among study subjects with W/M or M/M genotypes of GSTP1 and P53. Humans with null genotypes of GST P1 and P53 have been considered to be a higher risk group of carotid atherosclerosis due to their GSH

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deficiency. These findings derived from the study have given important evidences of atherogeneicity of arsenic. In addition, the joint effect between arsenic exposure and human susceptible genes on carotid atherosclerosis has also been found in this study. The hypothesis of this study has been well tested and proved.

3. References

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arsenic and prevalence of diabetes mellitus.

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and Chen, J. S. (1961) A clinical study of blackfoot disease in Taiwan: An endemic peripheral vascular disease. Memoirs College Med. Natl. Taiwan Univ., 7:1-17.

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& Sons Inc, New York.

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Table 1. Sociodemographic characteristics, life style and disease status of carotid atherosclerosis patients and controls

Variable Case Control OR (95%CI)^a OR (95%CI)^b

No(%) No(%)

Age

<55 25(8.9) 85(26.2) 1.0

55-64.9 105(37.5) 153(47.1) 2.3(1.4-3.9) ^

65+ 150(53.6) 87(26.7) 5.9(3.5-9.8) ^

Sex

Woman 127(45.4) 189(58.2) 1.0 1.0

Man 153(54.6) 136(41.8) 1.7(1.2-2.3) ^ 1.4(1.0-1.9)^§

BMI (kg/m²)

<20 47(17.4) 51(16.3) 1.0 1.0

20-25.9 127(47.0) 134(42.8) 1.0(0.6-1.6) 1.3(0.8-2.2)

26+ 96(35.6) 127(40.8) 0.8(0.5-1.3) 1.3(0.8-2.4)

Cigaratte smoking

No 157(56.1） 223（68.6） 1.0 1.0

Yes 123(43.9） 102（31.4） 1.7（1.2-2.4）^ 1.1（0.6-1.9）

Alcohol drinking

No 215(76.8） 276（85.2） 1.0 1.0

Yes 65(23.2） 48（14.8） 1.7（1.2-2.6）^ 1.3（0.8-2.1）

Hypertension

No 149(76.4) 243（87.7） 1.0 1.0

Yes 46(23.6） 34（12.3） 2.2（1.4-3.6）^ 2.4（1.4-4.0）^

Diabetes Mellitus

No 179(91.8） 255（92.1） 1.0 1.0

Yes 16(8.2） 22（7.9） 1.0（0.5-2.0） 1.1（0.5-2.2）

a: crude odds ratio b:age-sex-adjusted odds ratio §: 0.05<p<0.1  :0.01<p<0.05 :

0.001<p<0.01  :p<0.001

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Table 2 Age-sex-adjusted odds ratio (OR) and 95% confidence interval (CI) of carotid atherosclerosis by various arsenic exposure indices

No(%) No(%)

Arsenic

concentration in well water（g/L）

50 50（17.9） 115（35.4） 1.0 1.00

50.1-99.9 92（32.9） 84（25.8） 2.5（1.6-3.9）^ 2.13（1.04-4.32）^

100+ 138（49.2） 126（38.8） 2.5（1.02-4.02）^ 2.13（1.04-4.32）^

Cumulative arsenic exposure

(mg/L-year）

<1.0 58（20.7） 101（31.1） 1.0 1.0

1.0-6.9 150（53.6） 156（48.0） 1.7（1.1-2.5）^ 1.8（1.2-2.8）^

7.0+ 72（25.7） 68（20.9） 1.8（1.2-2.9）^ 1.9（1.1-3.0）^

Duration of drinking well water（year）

<30 52（18.6） 57（71.03） 1.0 1.0

30-49 127（45.4） 167（47.1） 0.8（0.5-1.3） 0.9（0.5-1.4）

50+ 101（36.0） 101（33.1） 1.1（0.7-1.7） 0.8（0.5-1.4）

a: crude odds ratio b:age-sex-adjusted odds ratio

§: 0.05<p<0.1  :0.01<p<0.05 : 0.001<p<0.01  :p<0.001

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Table 3 Age-sex-adjusted odds ratio (OR) and 95% confidence interval (CI) of carotid atherosclerosis by genetic polymorphisms of GSTM1, T1 , P1 and P53

No(%) No(%)

GSTM1

Non-null 136（48.7） 139（42.8） 1.0 1.0

Null 143（51.3） 186（57.2） 0.8（0.6-1.1） 0.9（0.5-1.0）

GSTT1

Non-null 134（48.0） 133（40.9） 1.0 1.0

Null 145（52.0） 192（59.1） 0.8（0.5-1.0）^§ 0.7（0.5-1.0）^§

GSTP1

W/W 178（63.8） 248（76.4） 1.0 1.0

W/M 95（34.1） 71（21.8） 1.8（1.3-2.6）^ 2.0（1.4-3.0）^

M/M 6（2.1） 6（1.8）

P53

W/W 53（19） 91（28） 1.0 1.0

W/M 175（62.7） 175（53.8） 1.7（1.1-2.4）^ 1.9（1.3-2.9）^

M/M 51（18.3） 59（18.2）

a: crude odds ratio b:age-sex-adjusted odds ratio

§: 0.05<p<0.1  :0.01<p<0.05 : 0.001<p<0.01  :p<0.001

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Table 4 Age-sex-adjusted odds ratio (OR) and 95% confidence interval (CI) of carotid atherosclerosis by genetic polymorphisms of GSTP1,and various arsenic exposure indices

GSTP1

Variable W/W W/M or M/M

case control OR^a(95%CI) case control OR^a(95%CI) Arsenic concentration

in well water（g/L）

50 29 85 1.0 20 31 1.0

50.1-99.9 60 66 2.7(1.5-4.9)^ 32 18 4.1(1.-10.0)^

100+ 89 97 2.7(1.6-4.5)^ 49 29 3.2(1.4-7.1)^

Cumulative arsenic exposure（mg/L-year）

<1.0 39 78 1.0 18 24 1.0

1.0-6.9 93 120 1.6(1.0-2.6)^§ 57 36 2.4(1.1-5.4)^

7.0+ 46 50 1.7(1.0-3.1)^§ 26 18 2.1(0.8-5.1)

<30 33 43 1.0 19 14 1.0

30-49 84 126 0.9(0.5-1.5) 43 41 0.8(0.4-2.0)

50+ 61 79 0.7(0.4-2.6) 39 23 1.0(0.4-2.6)

a: age-sex-adjusted odds ratio

§: 0.05<p<0.1  :0.01<p<0.05 : 0.001<p<0.01  :p<0.001 Wild/Wild: W/W; Wild/Mutant or Mutant/Mutant: W/M or M/M

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Table 5 Age-sex-adjusted odds ratio (OR) and 95% confidence interval (CI) of carotid atherosclerosis by genetic polymorphisms of P53,and various arsenic exposure indices

P53

Variable W/W W/M or M/M

case control OR^a(95%CI) case control OR^a(95%CI) Arsenic concentration in

well water（g/L）

50 19 40 1.0 30 75 1.0

50.1-99.9 15 26 1.3(0.5-3.0) 77 58 3.8(2.1-6.8)^

100+ 19 25 1.5(0.7-3.5) 119 101 3.1(1.8-5.3)^

Cumulative arsenic exposure（mg/L-year）

<1.0 18 35 1.0 39 67 1.0

1.0-6.9 24 40 1.2(0.5-2.5) 126 115 2.1(1.2-3.4)^

7.0+ 11 16 1.3(0.5-3.3) 61 52 2.1(1.2-3.8)^

<30 10 15 1.0 42 42 1.0