Effects of arsenic exposure and genetic polymorphisms of p53,
glutathione S-transferase M1, T1, and P1 on the risk of
carotid atherosclerosis in Taiwan
Yuan-Hung Wang
a, Meei-Maan Wu
a, Chi-Tzong Hong
b, Li-Ming Lien
c, Yi-Chen Hsieh
a,
Hung-Pin Tseng
d, Shu-Feng Chang
e, Che-Long Su
e, Hung-Yi Chiou
a,∗, Chien-Jen Chen
faSchool of Public Health, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan bDepartment of Neurology, Taipei Medical University-Wan Fang Hospital, Taipei 110, Taiwan cDepartment of Neurology, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan dDepartment of Neurology, Lo-Hsu Foundation, Lotung Poh-Ai Hospital, I-Lan 265, Taiwan
eDepartment of Neurology, Ming Jong Hospital, Pingtung 900, Taiwan
fGraduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei 100, Taiwan
Received 30 September 2005; received in revised form 25 May 2006; accepted 25 July 2006 Available online 14 September 2006
Abstract
To evaluate the joint effects between genetic polymorphisms of glutathione S-transferase M1, T1, P1, and p53, and arsenic exposure through drinking well water on the risk of carotid atherosclerosis, 605 residents including 289 men and 316 women were recruited from a northeastern area of Taiwan. Carotid atherosclerosis was diagnosed by either a carotid artery intima-media thickness (IMT) of >1.0 mm, a plaque score of ≥1, or stenosis of >50%. A significant age- and gender-adjusted odds ratio of 3.3 for the development of carotid atherosclerosis was observed among the high-arsenic exposure group who drank well water containing arsenic at levels >50g/L. The high-arsenic exposure group with GSTP1 variant genotypes of Ile/Val and Val/Val, and with the p53 variant genotypes of Arg/Pro and Pro/Pro had 6.0- and 3.1-fold higher risks of carotid atherosclerosis, respectively. In addition, the high-arsenic exposure group with one or two variant genotypes of GSTP1 and p53 had 2.8- and 6.1-fold higher risks of carotid atherosclerosis, respectively, and showed a dose-dependent relationship. A multivariate-adjusted odds ratio of 3.4 for the risk of carotid atherosclerosis among study subjects with the two variant genotypes of GSTP1 and p53 was also found. Our study showed the joint effects on the risk of carotid atherosclerosis between the genetic polymorphisms of GSTP1 and p53, and arsenic exposure.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Arsenic; Glutathione S-transferase; p53; Atherosclerosis
1. Introduction
Arsenic is widely distributed in nature and mainly trans-ported by water. Humans are exposed to arsenic through water, air, food, and medication, mainly by the way of ingestion and inhalation and rarely through skin absorption. In many parts of the world, including Taiwan, Argentina, Chile, Bangladesh, and China, it is a natural contaminant
∗Corresponding author. Tel.: +886 2 23779189; fax: +886 2 23779188.
E-mail address:hychiou@tmu.edu.tw(H.-Y. Chiou).
of ground water [1]. It can also be found in relatively high amounts in an organic form in seafoods, but this is easily excreted and is considered nontoxic[2]. The setting of the maximum contaminant level (MCL) that determines the concentration of arsenic in public water supplies has been an extraordinarily protracted process in the US. Recently, the MCL was lowered to 10g/L from the 50 g/L standard established in 1942[3]. Inorganic arsenic has been well doc-umented as one of the major risk factors for blackfoot disease (BFD), a unique peripheral vascular disease identified in the endemic area of arseniasis located on the southwestern 0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
coast of Taiwan where residents used high-arsenic artesian well water for more than 50 years [4]. The pathological types of BFD include arteriosclerosis obliterans (70%) and thromboangiitis obliterans (30%) which develop from severe underlying systemic arteriosclerosis[5]. Current studies have also reported that long-term exposure to arsenic in drinking water is significantly associated with the risk of ischemic heart disease, cerebrovascular disease, and peripheral vascular disease in Taiwan[6–8]. In addition, associations of arsenic with hypertension and diabetes mellitus have also been reported in Taiwan and Bangladesh[9–11].
Carotid atherosclerosis consists of subclinical lesions of the superficial carotid artery, which has been recognized as an independent risk factor of arterial diseases that lead to car-diac and cerebral infarction. An increase in the intima-media thickness (IMT) of the carotid artery, which is caused by the accumulation of cells, lipids, and connective tissue, may reflect earlier changes. In addition, stenosis and plaque for-mation are relatively late manifestations of the atherosclerotic process[12,13]. Both human and animal studies have found that increased apoptosis occurs in atherosclerotic lesions, which mediate tissue turnover and lesion development. The tumor-suppressive gene, p53, has been found in vascular cells and may participate in the regulation of vascular apoptosis during the development of atherosclerosis[14,15].
The main arsenic species in artesian well water is arse-nate. The mechanisms of arsenic metabolism and excretion in humans are not well elucidated. A substantial fraction of arsenate, which is readily absorbed from the gastrointestinal tract by humans, is reduced in the blood to arsenite. Arsenite is methylated mainly in the liver to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). The methylation is believed to occur via alternating reduction of pentava-lent arsenic (AsV) to trivalent arsenic (AsIII) and addition of a methyl group. S-Adenosylmethionine (SAM) is the main methyl donor in arsenic methylation. Because AsIII is more toxic than AsV, the initial step in the biotransformation of arsenate may be regarded as a bioactivation. However, much of the formed AsIII is distributed to the tissues where it is methylated to MMA and DMA[16–18]. Glutathione (GSH) and probably other thiols serve as reducing agents for AsV and MMAV[19]. According to the proposed mechanism of arsenic methylation, the biomethylation of inorganic arsenic (InAs) generates mono- and dimethyl arsenic species, which are more readily excreted than InAs. Thus, the methylation of InAs has traditionally been considered to be a detoxifica-tion pathway. However, a growing literature of experimental studies indicates that the trivalent methylated arsenic inter-mediates (MMAIII and DMAIII) may be more toxic than InAsIIIor any of the pentavalent intermediates[20,21]. Glu-tathione S-transferases (GSTs) are a large family of phase II detoxification enzymes that catalyze the conjugation of GSH to a wide range of hydrophobic and electrophilic com-pounds[22]. Based on sequence homology and immunologi-cal crossreactivity, human cytosolic GSTs have been grouped into seven families, designated GST Alpha, Mu, Pi, Sigma,
Omega, Theta, and Zeta[23]. Humans with null genotypes of GSTM1, T1 and the variant genotypes of GSTP1 are consid-ered to be a high-risk group for cancers due to their deficiency in GSTs. The aim of this study was to evaluate the joint effects of arsenic exposure through drinking water and genetic poly-morphisms of GSTM1, T1, P1, and p53 on the risk of carotid atherosclerosis.
2. Methods
2.1. Study area and subjects
Subjects were recruited from the Lanyang Basin of Ilan County in northeastern Taiwan. During the years of 1991–1994, a total of 8088 residents aged≥40 years from 18 villages in four townships were interviewed and included as the study cohort[8]. In the year of 1996, we had completed home-visit follow-up interview and had sent invitation let-ter to 5146 subjects out of the original cohort for attending a health examination held in 1997–1998. Finally, a total of 1318 cohort members (≥40 years) who agreed to participate in this study and finished the carotid ultrasound assessment of extracranial carotid artery (ECCA) being conducted in Lotung Poh-Ai hospital. Case patients were diagnosed as having prevalent asymptomatic carotid atherosclerosis by a neurologist based on either a mean carotid IMT of >1.0 mm, plaque occurrence in at least two locations on one side (right or left) or the occurrence of stenosis of >50% in the left or right CCA. Among these examinees, a total of 605 sub-jects (including 279 cases and 326 controls) were randomly selected without any matched factors. These study subjects were not substantially different from the original cohort. This study was approved by the institutional review board for human subjects of Taipei Medical University, Taipei, Taiwan and each subject provided written informed consent. 2.2. Questionnaire interview and determination of arsenic in the well water
A standardized personal interview was conducted by well-trained public health nurses based on a structured ques-tionnaire. Information of the personal interview included history of well water consumption, residential history, sociodemographic characteristics, levels of cigarette smok-ing and alcohol consumption, physical activities, as well as personal and familial history of hypertension, diabetes mellitus, cerebrovascular disease, heart disease, and cancer of various organs. Well water samples were collected during the home interview, immediately acidified with hydrochloric acid, and then stored at−20◦C until the subsequent assay. Hydride generation combined with flame atomic absorption spectrometry was used to determine the arsenic concentra-tion in these samples. The standard calibraconcentra-tion curve was established by serial dilution of standard arsenic solution as 1, 2, 4, 6, 8, 10, 20, 30, 40 and 50 ppb. The correlation coefficient of the standard calibration curve is between 0.996
and 0.999. Recovery rate ranged from 95% to 106%. The arsenic concentration in well water was found to range from undetectable (<0.15g/L) to 3.59 × 103g/L. The various exposure indices of arsenic through drinking water were described in a previous study[24].
2.3. Ultrasound measurements of carotid atherosclerosis To evaluate the extent of carotid atherosclerosis, the ECCA was scanned bilaterally with a Hewlett-Packard Sono1000 ultrasound system, equipped with 7.5 MHz frequency in the B-mode and 5.6 MHz frequency in the pulsed-Doppler mode. The duplex scanning and evaluation of study subjects were described in a previous study [25]. Indications of carotid atherosclerosis were assessed mainly based on three indices: (1) the ECCA intima-media thickness (IMT); (2) the ECCA plaque score and (3) the maximal level of ECCA stenosis. The plaque score was also used as an index of atherosclero-sis. The plaque score was counted if any plaque was observed in the areas of the external carotid artery (ECA), internal carotid artery (ICA), carotid bulb, or CCA. Study subjects were diagnosed as having prevalent asymptomatic carotid atherosclerosis by a neurologist based on a mean carotid IMT of >1.0 mm and either plaque occurrence in at least two loca-tions on one side (right or left) or the occurrence of stenosis of >50% in the left or right CCA.
2.4. Biospecimen collection and laboratory measurements
Ten milliliters of whole-blood samples was collected from study subjects using a disposable vacuum syringe and needle, centrifuged to separate the buffy coat and plasma, and stored at−70◦C until the subsequent assay. DNA was purified from the buffy coat by the Viogene Blood and Tissue Genomic DNA Miniprep System (Viogene Inc., Taipei, Taiwan) resus-pended in deionized distilled water, and stored at−20◦C until genotyping. The primers of GSTM1 were G5 (5-GAA CTC CCT GAA AAG CTA AAG C-3) and G6 (5-GTT GGG CTC AAA TAT ACG GTG G-3), and the PCR product showed a 215 bp band in the non-null genotype. The primers of GSTT1 were T1F (5-TTC CTT ACT GGT CCT CAC ATC TC-3) and T1R (5-TCA CCG GAT CAT GGC CAG CA-3), and the PCR product showed a 480 bp band in the non-null genotype. For GSTP1 and p53, PCR-RFLP (restriction fragment length polymorphism) was carried out with PCR primers P105F (5-ACC CCA GGG CTC TAT GGG AA-3) and P105R (5 -TGA GGG CAC AAG AAG CCC CT-3) for GSTP1 and p53-1 (5-ATC TAC AGT CCC CCT TGC CG-3) and p53-2 (5-GCA ACT GAC CGT GCA AGT CA-3) for p53, respec-tively. The GSTP1 PCR product (176 bp) was subjected to BsmAI digestion overnight at 55◦C, then loaded onto a 3% agarose gel and electrophoresed. The Ile/Ile genotype (wild type) showed a single band (176 bp), the Val/Val genotype (homozygote variant type) showed two bands (91 and 85 bp), and the Ile/Val genotype (heterozygote variant type) showed
three bands (176, 91, and 85 bp). The p53 PCR product (296 bp) was subjected to BstUI digestion overnight at 60◦C, then loaded onto a 3% agarose gel and electrophoresed. The Pro/Pro genotype (homozygote variant type) showed a single band (296 bp), the Arg/Arg genotype (wild type) showed two bands (169 and 127 bp), and the Arg/Pro genotype (heterozy-gote variant type) showed three bands (296, 169, and 127 bp). Beta-globulin and non-DNA template were used as internal positive and negative controls, respectively, in each experi-ment, and 10% of the total samples were randomly selected and reanalyzed with 100% of concordance.
2.5. Statistical analysis
Univariate analysis was conducted to test for risk fac-tors including age, gender, body mass index (BMI), cigarette smoking, alcohol consumption, total cholesterol, triglyc-eride, hypertension, diabetes mellitus and arsenic concen-tration in well water. To evaluate the associations between these risk factors and the prevalence of carotid atheroscle-rosis, a multiple logistic regression analysis was used to estimate the age- and gender-adjusted odds ratio (OR) and 95% confidence interval (95% CI). The statistical signifi-cance of each multivariate-adjusted odds ratio was examined by significance testing of the regression coefficient based on the maximum likelihood method. Tests for Hardy–Weinberg equilibrium were conducted by comparing observed geno-type frequencies with expected genogeno-type frequencies among control subjects using a2-test. Based on our initial results from univariate analysis, several variables such as age, gen-der, BMI, cigarette smoking, alcohol consumption, hyper-tension, diabetes mellitus, total cholesterol, triglyceride and arsenic concentration in well water were included in the sub-sequent analyses as shown in models I and II to control for potential confounding effect caused by these variables. Sta-tistical Analysis Software (SAS Version 6.12; SAS Institute, Cary, NC) was applied to all statistical analyses. Values of p < 0.05 were considered statistically significant.
3. Results
In total, 605 study subjects, including 279 carotid atherosclerosis patients and 326 controls, were recruited in this study. Among them, there were 289 men and 316 women. As shown inTable 1, compared with the youngest age group, significant odds ratios of 5.8 and 2.3 were observed for the groups aged ≥65 and between 55 and 64.9 years old, respectively. Men had a higher risk of carotid atheroscle-rosis than women. A significant risk for development of carotid atherosclerosis was also observed among hyperten-sive patients with an age- and gender-adjusted odds ratio of 2.4. Compared with subjects whose BMI was≤24 as a ref-erence group, subjects with a BMI of >24 had a significant 1.7-fold higher risk of carotid atherosclerosis after adjust-ing for age and gender. Subjects with higher total cholesterol
Table 1
Characteristics of subjects with carotid atherosclerosis and of controls
Casesa(n = 279) Controls (n = 326) OR (95% CI)b OR (95% CI)c Age (years) <55 25 (9.0%) 85 (26.1%) 1.0 55–64.9 105 (37.6%) 153 (46.9%) 2.3 (1.4–3.9)** ≥65 149 (53.4%) 88 (27.0%) 5.8 (3.4–9.7)*** Gender Female 127 (45.5%) 190 (58.2%) 1.0 1.0 Male 152 (54.5%) 136 (41.8%) 1.7 (1.2–2.3)*** 1.4 (1.0–1.9) BMI (kg/m2) ≤24 174 (62.4%) 232 (71.2%) 1.0 1.0 >24 105 (37.6%) 94 (28.8%) 1.5 (1.1–2.1)* 1.7 (1.2–2.5)** Cigarette smoking No 157 (56.3%) 223 (68.4%) 1.0 1.0 Yes 122 (43.7%) 103 (31.6%) 1.7 (1.2–2.4)** 1.1 (0.6–1.8) Alcohol consumption No 214 (76.7%) 278 (85.3%) 1.0 1.0 Yes 65 (23.3%) 48 (14.7%) 1.7 (1.2–2.6)** 1.3 (0.8–2.1) Hypertensiond No 148 (76.3%) 244 (87.8%) 1.0 1.0 Yes 46 (23.7%) 34 (12.2%) 2.2 (1.4–3.6)** 2.4 (1.5–4.1)** Diabetes mellitusd No 178 (91.8%) 256 (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) Total cholesterol (mg/dL) <200 166 (59.5%) 225 (69.1%) 1.0 1.0 ≥200 113 (40.5%) 101 (30.9%) 1.5 (1.1–2.1)* 1.6 (1.1–2.3)** Triglyceride (mg/dL) <200 247 (88.5%) 300 (92.0%) 1.0 1.0 ≥200 32 (11.5%) 26 (8.0%) 1.5 (0.9–2.6) 1.4 (0.8–2.4)
Arsenic concentration in well water (g/L)
≤10 19 (6.8%) 48 (14.7%) 1.0 1.0
10.1–50.0 30 (10.8%) 68 (20.9%) 1.1 (0.6–2.2) 1.4 (0.7–2.8)
≥50.1 230 (82.4%) 210 (64.4%) 2.8 (1.6–4.9)*** 3.3 (1.8–6.0)***
*0.01 < p < 0.05;**0.001 < p < 0.01;***p < 0.001. aCases: subjects with carotid atherosclerosis. b Crude odds ratio.
cAge- and gender-adjusted odds ratio; 95% CI: 95% confidence interval.
d Some subjects with no information about the history of hypertension and diabetes mellitus.
(≥200 mg/dL) had a significant 1.6-fold higher risk of carotid atherosclerosis after adjustment for age and gender. In addi-tion, smokers, alcohol drinkers, diabetes mellitus patients and subjects with higher triglyceride also had higher but not significant risks for the development of carotid atheroscle-rosis. A significant age- and gender-adjusted odds ratio of a 3.3-fold higher risk of carotid atherosclerosis was observed in the exposure group with an arsenic concentration in the well water of >50g/L. The genotype frequencies were in Hardy–Weinberg equilibrium in the control groups for both GSTP1 and p53 (p = 0.63 and 0.87, respectively). As illus-trated in Table 2, a significantly higher age- and gender-adjusted odds ratio of 2.0 for the development of carotid atherosclerosis was observed among study subjects with the Ile/Val and Val/Val genotypes of GSTP1 and of 1.9 for study subjects with the Arg/Pro and Pro/Pro genotypes of p53. In a gene–gene interaction analysis, compared with the reference
group (wild genotypes of GSTP1 and p53), those who car-ried one and two variant genotypes of GSTP1 and p53 had significant age- and gender-adjusted odds ratios of 1.8 and 3.8, respectively, for the development of carotid atheroscle-rosis. In a further trend test, it also showed a dose–response relationship. However, study subjects with the null genotype for GSTM1 and T1 did not have a higher risk of carotid atherosclerosis.
Table 3 shows that study subjects with the Ile/Val and Val/Val genotypes of GSTP1 had significantly higher age-and gender-adjusted odds ratios of 2.7 and 6.0 for the develop-ment of carotid atherosclerosis among those who drank well water containing arsenic concentrations of >50g/L. Study subjects with the Arg/Pro and Pro/Pro genotypes of p53 had a significantly higher age- and gender-adjusted odds ratio of 3.1 for the risk of carotid atherosclerosis among those individuals drinking well water containing arsenic >50g/L. As shown
Table 2
Age- and gender-adjusted odds ratio and 95% CI of subjects with carotid atherosclerosis and of controls by the genotypes of GSTM1, T1, P1 and p53 Casesa(n = 279) Controls (n = 325)b OR (95% CI)c OR (95% CI)d
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 Ile/Ile 178 (63.8%) 248 (76.4%) 1.0 1.0
Ile/Val and Val/Val 101 (36.2%) 77 (23.6%) 1.8 (1.3–2.6)*** 2.0 (1.4–3.0)***
p53
Arg/Arg 53 (19.0%) 91 (28.0%) 1.0 1.0
Arg/Pro and Pro/Pro 226 (81.0%) 234 (72.0%) 1.7 (1.1–2.4)* 1.9 (1.3–2.9)**
GSTP1and p53e
0 36 (12.9%) 67 (20.6%) 1.0 1.0
1 159 (57.0%) 205 (63.1%) 1.4 (0.9–2.3) 1.8 (1.1–2.9)*
2 84 (30.1%) 53 (16.3%) 2.9 (1.7–5.0)***, P for trend <0.0001 3.8 (2.2–6.7)***, P for trend <0.0001
*0.01 < p < 0.05;**0.001 < p < 0.01;***p < 0.001. aCase subjects with carotid atherosclerosis.
b For one control who lost the data of genotypes of GSTM1,T1,P1 and p53. cCrude odds ratio.
d Age- and gender-adjusted odds ratio; 95%CI: 95% confidence interval.
eTotal number of variant (homo- and hetero-zygote) genotypes of GSTP1 and p53.
inTable 4, significantly higher risks of 2.8 and 6.1 for the development of carotid atherosclerosis were observed among study subjects who carried one and two variant genotypes of GSTP1 and p53 and who had arsenic exposure through drink-ing well water with arsenic levels of >50g/L, thus showing a biological relationship.
Results of the multivariate models (models I and II) are shown inTable 5. Significant multivariate-adjusted risk fac-tors for the development of carotid atherosclerosis were age, hypertension, variant genotypes of GSTP1 and p53, and arsenic concentration in well water in model I. As illustrated in model II, the joint effects of variant genotypes of GSTP1 and p53 on the risk of carotid atherosclerosis were observed
Table 3
Age- and gender-adjusted odds ratio and 95% CI of subjects with carotid atherosclerosis and of controls by the genotypes of GSTP1, p53 and arsenic exposure indices
Arsenic concentration in well water (g/L)
≤50 >50
ORa 95% CI ORa 95% CI GSTP1
Ile/Ile 1.0 2.7 1.6–4.5**
Ile/Val and Val/Val 1.9 0.9–4.0 6.0 3.3–10.7***
p53
Arg/Arg 1.0 1.4 0.7–3.0
Arg/Pro and Pro/Pro 0.9 0.5–1.9 3.1 1.7–5.7*** *0.01 < p < 0.05;**0.001 < p < 0.01; ***p < 0.001.
aAge- and gender-adjusted odds ratio with arsenic exposure≤50 g/L
and GSTP1 genotype of Ile/Ile or p53 genotype of Arg/Arg.
Table 4
Age- and gender-adjusted odds ratio and 95% CI of subjects with carotid atherosclerosis and of controls by the genotypes of GSTP1, p53 and arsenic exposure indices
Number of variant genotype of GSTP1 and p53a
Arsenic concentration in well water (g/L)
≤50 >50 ORb 95% CI ORb 95% CI 0 1.0 1.4 0.6–3.3 1 1.0 0.4–2.2 2.8 1.3–6.0** 2 1.9 0.7–5.3 6.1 2.7–13.9*** *0.01 < p < 0.05;**0.001 < p < 0.01;***p < 0.001.
aTotal number of variant (homo- and hetero-zygote) genotypes of GSTP1
and p53.
bAge- and gender-adjusted odds ratio with arsenic exposure≤50 g/L
and GSTP1 genotype of Ile/Ile or p53 genotype of Arg/Arg.
as significantly independent risk factors after adjustment for those risk factors found in model I.
4. Discussion
The atherogenic effects of ingesting inorganic arsenic through drinking water have been well documented. Several studies in Taiwan have reported significant associations between long-term exposure to arsenic in drinking water and the risk for the development of atherosclerotic vascular diseases including ischemic heart disease, cerebrovascular disease, and peripheral vascular disease, all of which
Table 5
Multiple logistic regression analysis of related risk factors of subjects with carotid atherosclerosis and of controls
Variables Model I (OR (95% CI)a) Model II (OR (95% CI)a) Age <55 1.0 1.0 55–64.9 2.2 (1.3–3.7)** 2.2 (1.3–3.7)** ≥65 5.8 (3.3–10.2)*** 5.7 (3.3–10.0)*** Gender Female 1.0 1.0 Male 1.5 (0.9–2.7) 1.5 (0.9–2.7) BMI (kg/m2) ≤24 1.0 1.0 >24 1.2 (0.8–1.8) 1.2 (0.8–1.8) Cigarette smoking No 1.0 1.0 Yes 1.1 (0.6–1.8) 1.1 (0.6–1.8) Alcohol consumption No 1.0 1.0 Yes 1.3 (0.7–2.1) 1.3 (0.7–2.1) Hypertension No 1.0 1.0 Yes 1.8 (1.0–3.1)* 1.8 (1.0–3.0)* Diabetes mellitus No 1.0 1.0 Yes 0.8 (0.4–1.6) 0.7 (0.4–1.6) Total cholesterol (mg/dL) <200 1.0 1.0 ≥200 1.2 (0.8–1.7) 1.2 (0.8–1.7) Triglyceride (mg/dL) <200 1.0 1.0 ≥200 1.0 (0.5–1.9) 1.0 (0.5–1.9)
Arsenic concentration in well water (g/L)
≤10 1.0 1.0
10.1–50.0 1.6 (0.7–3.3) 1.6 (0.7–3.3) ≥50.1 3.0 (1.6–5.9)** 2.9 (1.5–5.7)**
GSTP1
Ile/Ile 1.0
Ile/Val and Val/Val 2.1 (1.4–3.1)*** p53
Arg/Arg 1.0
Arg/Pro and Pro/Pro 1.6 (1.1–2.5)*
Number of variant genotype of GSTP1 and p53b
0 1.0
1 1.6 (1.0–2.7)*
2 3.4 (1.9–6.2)***, P
for trend < 0.0001
*0.01 < p <0.05;**0.001 < p < 0.01;***p < 0.001.
aMultiple related risk factors-adjusted odds ratio; 95% CI: 95%
confi-dence interval.
b Total number of variant (homo- and hetero-zygote) genotypes of GSTP1
and p53.
showed dose–response relationships[25,26]. In addition, a significant dose–response relationship between long-term exposure to arsenic through drinking well water and risks of hypertension and diabetes mellitus was also found in Taiwan
[10,11]. Atherosclerosis combined with hypertension and diabetes mellitus predisposed residents to the development of stroke and ischemic heart disease which are the leading causes of death in developed countries[27]. In our current study which was based on personal exposure data carried out in the Lanyang Basin of northeastern Taiwan, compared with the reference group of arsenic exposure in well water at ≤10 g/L, there was a significant age- and gender-adjusted odds ratio of developing carotid atherosclerosis of 3.3 for the highest arsenic exposure group with arsenic concentrations in well water of >50g/L. Furthermore, an increased risk of atherosclerosis was also found among subjects with arsenic exposure through drinking well water containing arsenic concentrations which ranged between 10 and ≤50 g/L.
Age was significantly associated with the risk of carotid atherosclerosis. This implies that age is a major risk factor for carotid atherosclerosis. Subjects with a BMI of >24 had an age- and gender-adjusted odds ratio of a 1.7-fold higher risk of carotid atherosclerosis, compared with the reference group with a BMI of≤24. Our finding that hypertension was significantly associated with atherosclerosis risk is consis-tent with previous reports. However, a non-significant higher risk (OR = 1.1) of carotid atherosclerosis was observed for patients with diabetes mellitus. Besides, our results indicated that subjects with higher total cholesterol (≥200 mg/dL) had a significant age- and gender-adjusted odds ratio of 1.6. This result was concordant with the general concept that total cholesterol is a traditional risk factor of carotid atheroscle-rosis. However, cigarette smoking, alcohol consumption and higher triglyceride produced non-significant increased risks of carotid atherosclerosis, showing age- and gender-adjusted risks of 1.1, 1.3 and 1.4, respectively.
The concept that oxidized lipoproteins are involved in atherosclerotic lesion development was formulated from demonstrations that low-density lipoproteins can injure cells under certain conditions, which were later shown to be facil-itated oxidation of lipoproteins[28]. Based on this evidence, arsenic might be involved in the formulation of atherosclero-sis because it can induce oxidative stress which increases the level of accumulation of oxidized lipoproteins[29]. A recent study indicated that p53 accumulates in human atheroscle-rotic tissue in plaque areas with signs of chronic inflammation [30]. The effect of sodium arsenite on p53 protein levels may be relevant to the development of cancer, since its induction may reflect the presence of alterations in the cell proliferation ‘machinery’[31]. Inorganic arsenic is metabolized through methylation using SAM as the methyl donor and a methyl-transferase (MTase). Therefore, arsenic may be involved in alterations of MTase/SAM-dependent DNA methylation of the p53 tumor suppressor gene[32]. In this study, a signifi-cantly increased risk of carotid atherosclerosis was observed among study subjects with Arg/Pro and Pro/Pro genotypes of p53, with an age- and gender-adjusted odds ratio of 1.9. In addition, a highly significant synergistic effect on the risk of carotid atherosclerosis between these genotypes of p53 and
arsenic exposure of >50g/L was reported with an age- and gender-adjusted odds ratio of 3.1.
GSTs are necessary for arsenic methylation, perhaps through the formation of arsenite which is the major arsenic form for methylation, or through conjugation with arsenic [33]. Humans exposed to arsenic with null genotypes of GSTM1, T1 and the variant genotypes of GSTP1 have been considered to be a high-risk group for carotid atherosclerosis due to their deficiency in GSTs for efficiently conjugating arsenic with methyl groups to form hydrophilic metabolites. In this study, there were no significant differences in the risks of carotid atherosclerosis among study subjects with the null genotypes of GSTM1 and GSTT1. However, a significantly increased risk for the development of carotid atheroscle-rosis was observed among study subjects with the Ile/Val and Val/Val genotypes of GSTP1, with an age- and gender-adjusted odds ratio of 2.0. Previous studies suggested that genetic polymorphisms of GSTP1 exon 5 (Ile to Val) and exon 6 (Ala to Val) have functional relevance to the GST gene product resulting in reduced enzyme activity. There-fore, individuals with these variant genotypes of GSTP1 may be at greater risk for carotid atherosclerosis due to decreased detoxification of inorganic arsenic. A significant joint effect of the Ile/Val and Val/Val genotypes of GSTP1 and high-arsenic exposure (>50g/L) on the risk of developing carotid atherosclerosis was found in this study, with an age- and gender-adjusted odds ratio of 6.0.
Our results showed that those with one or two variant genotypes of GSTP1 and p53 had significant age- and gender-adjusted odds ratios of 1.8 and 3.8, respectively, compared with wild genotypes of GSTP1 and p53 as a reference group. The risk of carotid atherosclerosis increased as the num-ber of variant genotypes of these two genes increased. Joint effects on the risk of carotid atherosclerosis among study subjects who drank well water containing arsenic at a con-centration >50g/L and with one and two of variant geno-types of these two genes were 2.8 and 6.1, respectively. In addition, after adjustment for several traditional risk factors including age, gender, BMI, cigarette smoking, alcohol con-sumption, hypertension, diabetes mellitus, total cholesterol, triglyceride and arsenic exposure, significant multivariate-adjusted odds ratios of 2.1 (p = 0.0002) and 1.6 (p = 0.028) were observed among those with variant genotypes of GSTP1 and p53, respectively, in model I. Significant risks of carotid atherosclerosis were also found in model II for study sub-jects with two variant genotypes of GSTP1 and p53, with multivariate-adjusted odds ratios of 1.6 and 3.4. Based on the results inTables 4 and 5, joint effects between arsenic exposure and variant genotypes of GSTP1 and p53 on the risk for development of carotid atherosclerosis were found in this study.
We did not include in our analysis the completed infor-mation about the familial history of stroke or cardiovascular disease and LDL, which might be a limitation to our results. Nonetheless, we collected the major risk factors including age gender, BMI, cigarette smoking, alcohol consumption,
hypertension, diabetes mellitus, total cholesterol, triglyceride and arsenic concentration in well water. These important variables were further included in the subsequent models to control for the confounding effect. Regarding the IMT defini-tion, the method of IMT assessment in the present study had been published[25]. It implied that the IMT definition used in our study to evaluate carotid atherosclerosis is appropriate. A detailed history of residential village water consumption, including water source and duration of consumption obtained from the questionnaire interview was used to estimate the arsenic exposure. Additionally, a total of 3901 well-water samples (one sample from each household) were collected during home interview and subsequently analyzed with a quality control procedure[8]. Compared with previous stud-ies conducted in the BFD-endemic area[6,25], we can more exactly estimate the individual arsenic exposure for study subjects. To avoid selection bias, study subjects were invited to a representative hospital (Lotung Poh-Ai hospital) and the neurologist performed carotid ultrasound assessments with blindness to the individual arsenic exposure. In the future, more detailed information including dietary issues and famil-ial history of stroke or cardiovascular diseases should be completely collected in a larger study.
Acknowledgements
This study was supported by grants from the National Science Council of Taiwan (NSC89-2314-B-038-060; NSC90-2320-B-038-044; NSC92-2321-B-038-011); Top-notch Stroke Research Center Grant, Ministry of Education; Center of Excellence for Clinical Trial and Research in Neu-rology Specialty.
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