Protective effects of plasma alpha-tocopherols on the risk of inorganic arsenic-related urothelial carcinoma.

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Protective effects of plasma alpha-tocopherols on the risk of inorganic

arsenic-related urothelial carcinoma

Chi-Jung Chung

a,1

, Yeong-Shiau Pu

b

, Ying-Ting Chen

c

, Chien-Tien Su

d

, Chia-Chang Wu

a,e

,

Horng-Sheng Shiue

f

, Chao-Yuan Huang

b,g

, Yu-Mei Hsueh

c,

⁎

a

School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan

b

Department of Urology, National Taiwan University Hospital, Taipei, Taiwan

c

Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

d

Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan

e_{Department of Urology, Taipei Medical Universtiy-Shuang Ho Hospital, Taipei, Taiwan} f

Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan

g

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

a b s t r a c t

a r t i c l e i n f o

Article history: Received 28 July 2010

Received in revised form 12 November 2010 Accepted 29 November 2010

Available online 11 January 2011 Keywords:

Urinary arsenic proﬁles Urothelial carcinoma 8-OHdG

Plasma micronutrients

Arsenic plays an important role in producing oxidative stress in cultured cells. To investigate the interaction between high oxidative stress and low arsenic methylation capacity on arsenic carcinogenesis, a case–control study was conducted to evaluate the relationship among the indices of oxidative stress, such as urinary 8-hydroxydeoxyquanine (8-OHdG), as well as plasma micronutrients and urinary arsenic profiles on urothelial carcinoma (UC) risk. Urinary 8-OHdG was measured using high-sensitivity enzyme-linked immunosorbent assay kits. The urinary arsenic species were analyzed using high-performance liquid chromatography and hydride generator-atomic absorption spectrometry. Plasma micronutrient levels were analyzed using reversed-phase high-performance liquid chromatography. The present study showed a significant protective effect of plasma alpha-tocopherol on UC risk. Plasma alpha-tocopherol levels were significantly inversely related to urinary total arsenic concentrations and inorganic arsenic percentage (InAs%), and significantly positively related to dimethylarsinic acid percentage (DMA%). There were no correlations between plasma micronutrients and urinary 8-OHdG. Study participants with lower alpha-tocopherol and higher urinary total arsenic, higher InAs%, higher MMA%, and lower DMA% had a higher UC risk than those with higher alpha-tocopherol and lower urinary total arsenic, lower InAs%, lower MMA%, and higher DMA%. These results suggest that plasma alpha-tocopherol might modify the risk of inorganic arsenic-related UC.

1. Introduction

Bladder cancer is the most common malignancy of the urinary tract and it was estimated that 1900 new cases would be diagnosed and 747 deaths would occur in Taiwan in 2005 according to the data from Department of Health, the Executive Yuan. Cigarette smoking and carcinogens in drinking water could have a direct effect on urinary tract cancer development (Negri and La, 2001). Inorganic arsenic is recognized as a potent human carcinogen. Epidemiological evidences showed that

exposure arsenic in drinking water were associated with skin cancer, liver cancer and bladder cancer (Chiou et al., 1995;Hsueh et al., 1997). Huang et al. reported a signi_{ﬁcant association between insufﬁcient} arsenic methylation and the development of urothelial carcinoma (UC) conducted from the cohort study in the arseniasis hyperendemic area (Huang et al., 2008). Further data showed that smokers in Argentina and the US with a high monomethylarsonic acid percentage (%MMA) had a high odds ratio of bladder cancer (Steinmaus et al., 2006).

Oxidative stress resulting from the disturbance of the pro-oxidant/ antioxidant balance might be related to oncogenic stimulation (Valko et al., 2006). DNA lesions induced by reactive oxygen species (ROS), including 8-hydroxydeoxyquanine (8-OHdG), could be involved in the etiology of cancer (Beevi et al., 2007). Urinary 8-OHdG is one of the most abundant markers of DNA damage, which is increased in cancer patients, compared to the general population (Howard et al., 1998). Plasma retinol, alpha-tocopherols, lycopene, and beta-carotenes are the major lipophilic antioxidant constituents of fruits and vegetables. Dietary intake of fruits and vegetables has been associated with increased levels of plasma lipophilic micronutrients in epidemiological Abbreviations: UC, urothelial carcinoma; InAs, inorganic arsenic; iAs3+

, inorganic arsenite; iAs5+

, inorganic arsenate; DMA5+

, dimethylarsinic acid; MMA5+

, mono-methylarsonic acid; % InAs, percentage of InAs; % DMA5+_{, percentage of DMA5+; %}

MMA5+_{, percentage of MMA5+.}

⁎ Corresponding author. Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: + 886 2 27361661x6513; fax: + 886 2 27384831.

E-mail address:ymhsueh@tmu.edu.tw(Y.-M. Hsueh).

1

Current address: Department of Medical Research, China Medical University Hospital, Taichung, Taiwan.

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Science of the Total Environment

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studies (Briviba et al., 2008). Exposure to antioxidant supplement or tomato extract could effectively attenuate oxidative stress, acute in_{flammation, tumor cell proliferation, cell cycle progression, and} induce the expression of apoptotic Bax protein in in-vitro studies (Amir et al., 1999;Jamshidzadeh et al., 2008;Tang et al., 2008). The extent of DNA damage could be altered by reducing the TNF-_α level, DNA-protein cross-links, DNA stand breaks, and inhibiting the activation of caspase cascade in rats treated with arsenic along with alpha-tocopherol (Ramanathan et al., 2005;Kadirvel et al., 2007). Until now, epidemiological studies evaluating the association between major lipophilic antioxidants and bladder cancer risk have reported conflicting findings (Zeegers et al., 2001;Michaud et al., 2002;Hung et al., 2006).

There is evidence that dietary supplementation could improve oxidative disturbances, and that plasma micronutrient levels could prevent cancer incidences (Woodside et al., 2005). However, the association between plasma micronutrients and arsenic-related DNA damage is still unclear (Chung et al., 2006; Hsueh et al., 1997; Verret et al., 2005). Therefore, the objective of the present study is to investigate whether plasma micronutrients could modify UC risk resulting from altered DNA damage associated with urinary arsenic concentrations.

2. Materials and methods 2.1. Study participants

We conducted a hospital-based case–control study. Study proto-cols have been described in detail elsewhere (Pu et al., 2007). Brie_fly, 170 UC cases and 402 healthy control participants were recruited from September 2002 to April 2006. All UC cases were diagnosed with histological con_{firmation and pathological verification. This was done} using routine urological practices, including endoscopic biopsy or surgical resection of urinary tract tumors, followed by histopatholog-ical examination by board-certified pathologists. Healthy control participants who had no prior history of cancer were matched to UC patients in terms of age ±5 years and gender. The majority of study participants (N80%) lived in Taipei city and drank tap water from Taipei Water Department of the Taipei City Government. The average arsenic concentration of tap water is 0.7μg/L, but ranges from non-detectable to 4.0μg/L. The Research Ethics Committee of National Taiwan University Hospital, Taipei, Taiwan, approved the study. All participants provided informed consent before the questionnaire, interview, and bio-specimen collection. The study was consistent with the World Medical Association Declaration of Helsinki.

2.2. Questionnaire, interview, and biological specimen collection Well-trained interviewers collected detailed information on demo-graphics and socioeconomic characteristics, lifestyle factors, such as cigarette smoking and alcohol consumption, residential and occupa-tional history, and personal and family histories of disease through a structured questionnaire. Spot urine samples were immediately transferred to a−20 °C freezer until required for urinary arsenic proﬁle and 8-OHdG analyses. In addition, blood specimens were centrifuged at 3000 rpm for 10 min to separate plasma and stored at−80 °C until micronutrient analyses.

2.3. Urinary arsenic proﬁles and 8-OHdG measurement Levels of urinary arsenic proﬁles including iAs3+

, iAs5+, MMA5+, and DMA5+_{were measured by high-performance liquid}

chromatog-raphy (HPLC) equipped with a hydride generator and atomic absorption spectrometer (HG-AAS) (Hsueh et al., 1998a). Standard reference material SRM 2670 urine was obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA),

which contains 480 ± 100μg/L of inorganic arsenic. SRM 2670 was used as a quality standard and analyzed with the urine samples. The mean value of arsenic in our SRM 2670 sample was 507 ± 17_{μg/L (n=4).} Recovery rates of the four arsenic species were calculated using the following formula: ([(sample spiked standard solution concentration)− sample concentration] / standard solution concentration × 100). Re-covery rates for iAs3+_{, DMA}5+_{, MMA}5+_{, and iAs}5+_{ranged from 93.8}

to 102.2%, with detection limits of 0.02, 0.08, 0.05, and 0.07μg/L, respectively. Urinary 8-OHdG was analyzed using a competitive enzyme-linked immunosorbent assay (ELISA) kit (Japan Institute for the Control of Aging, Fukuroi, Japan), described in detail elsewhere (Chung et al., 2008). The detection range of the ELISA assay was 0.5– 200 ng/mL and the intra-and inter-assay coefﬁcients of variance (CV) were 9.8% and 6.7%, respectively. Urinary arsenic species are stable at least 6 months when stored at−20 °C (Chen et al., 2002). Therefore, the urine assays of the urinary arsenic species and 8-OHdG were performed within 6 months after collection.

2.4. Determination of plasma micronutrient levels

Plasma concentrations of retinol, alpha-tocopherol, lycopene, and beta-carotene were analyzed using reversed-phase HPLC (Hitachi Inc, Tokyo, Japan) according to the protocol described previously (Hsueh et al., 1998b). The mobile phase concentration of methanol: acetonitrite:chloroform was 47:47:6, and multi-wavelength monitor-ing was carried out durmonitor-ing the experiment. Micronutrient levels were detected at 325 nm for retinol, 280 nm for alpha-tocopherol, and 466 nm for lycopene and beta-carotene. Plasma specimens for each case control set were thawed from a−80 °C refrigerator in dim light at room temperature and assayed on the same day to ensure that temporal variability in the laboratory assays would affect cases and controls equally. All laboratory personnel were unaware of the disease status of the subjects whose serum samples were tested. Recovery rates were 96.1% for retinol, 94.6% for alpha-tocopherol, 88.4% for

Table 1

Demographic characteristics and lifestyle for 170 UC cases and 402 controls.

Variables UC cases Control ORa

(95% CI) P value (n = 170) (n = 402)

Age (years) (mean± SE) 62.14 ± 1.08 61.50 ± 0.71 1.00 (0.99–1.02) 0.65 Gender Male 123 (72.35) 277 (68.91) 1.16 (0.78–1.73) 0.46 Female 47 (27.65) 125 (31.09) 1.00 Education Elementary school or below 66 (38.82) 72 (18.05) 1.00b High school 67 (39.41) 138 (34.59) 0.45 (0.29–0.72) b0.01 College or above 37 (21.76) 189 (47.37) 0.17 (0.10–0.29) b0.01 Paternal ethnicity Fukien Taiwanese 124 (72.94) 217 (54.11) 1.00b Hakka Taiwanese 16 (9.41) 45 (11.22) 0.65 (0.35–1.20) 0.17 Mainland Chinese 30 (17.65) 137 (34.16) 0.34 (0.21–0.55) b0.01 Cumulative cigarette smoking (pack-years) 17.71 ± 2.00 9.99 ± 0.97 1.02 (1.01–1.03) b0.01 0 78 (49.06) 255 (65.89) 1.00 N0 81 (50.94) 132 (34.11) 2.42 (1.53–3.82) b0.01 Alcohol drinking Never 99 (58.24) 206 (51.37) 1.00 Occasional 29 (17.06) 130 (32.42) 0.44 (0.27–0.71) b0.01 Regular 42 (24.71) 65 (16.21) 1.20 (0.73–1.96) 0.47 Pesticide usage No 142 (84.02) 386 (96.50) 1.00 Yes 27 (15.98) 14 (3.50) 5.25 (2.67–10.33) b0.01 SE: standard error.

a

Adjusted by age and gender.

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lycopene, and 99.4% for beta-carotene. Furthermore, we used alpha-tocopherol acetate as an internal control to minimize systematic error and the CV was_b5%.

2.5. Statistical analysis

All analyses were performed using SAS 9.0 (SAS, version 9.0, Cary, NC). Total arsenic concentration (μg/L) was the sum of iAs3+_{, iAs}5+_,

MMA5+, and DMA5+levels. The relative proportion of urinary arsenic species (iAs3+_{%, iAs}5+_{%, MMA}5+_{%, and DMA}5+_{%) was calculated}

by dividing each arsenic species level by the total arsenic concentra-tion. Inorganic arsenic percentage (InAs%) was the sum of iAs3+_%

and iAs5+_{%. Differences between cases and controls were tested by}

Student's t-test in continuous variables. A Log10-transformation was applied to the factors with an abnormal distribution before statistical analyses. A multiple linear regression model was used to estimate the effects of potential factors, including various arsenic species and urinary 8-OHdG, on plasma micronutrients. Tertile values of urinary arsenic profile, plasma micronutrient, and 8-OHdG levels of the con-trols were used as cutoff points for the trend test. Multiple logistic regression models were used to estimate the multivariate adjusted odds ratio (OR) and the 95% confidence interval (CI).Barr et al. (2005) reported that the significant predictors of urinary creatinine concen-trations include age, sex, race, body mass index and fat-free mass. In addition, one study of 1650 adults in Bangladesh revealed that urinary creatinine concentrations are significantly correlated with plasma folate concentrations, particularly among males (Gamble and Liu,

2005). Therefore, we treated urinary creatinine as a separate variable in the regression model. For the joint effect analysis, the cutoff points for total arsenic, plasma micronutrient, and 8-OHdG levels were the respective medians of the controls. Finally, we estimated both the additive model (synergy index) and the multiplicative model to evaluate the joint effects of plasma micronutrients, as well as various arsenic species, on UC risk. An observed synergy index value that departs substantially from the expected additive null (i.e., synergy index not equal to 1) suggests an additive interaction effect (Hosmer and Lemeshow, 1992). The binary interaction terms were calculated by multiplying the indicators for two explored risk factors and then added to the main effect models; the p values were also calculated. 3. Results

The sociodemographic characteristics of UC cases and controls are shown inTable 1. Participants who had higher educational levels and whose paternal ethnicity was Mainland Chinese had a significantly lower UC risk than those with lower educational levels and whose paternal ethnicity was Fukien Taiwanese. Occasional alcohol drinkers had a significantly lower UC risk than non-drinkers. Cigarette smokers and pesticide users had a significantly higher UC risk than non-smokers and non-users, respectively.

Mean ± standard error (SE) of urinary arsenic proﬁles, 8-OHdG, and plasma micronutrients stratiﬁed by UC status are shown in Table 2. With trend analysis on exposure strata in tertiles, high total arsenic, high MMA%, low DMA%, and low plasma alpha-tocopherol Table 2

Urinary arsenic proﬁles and 8-OHdG and plasma micronutrients concentrations of UC 170 cases and 402 controls.

Variables UC cases (n = 170) No. (%) Control (n = 402) No. (%)

Crude OR (95% CI) P value ORa_{(95% CI)} _{P value}

Total arsenic (μg/g creatinine) (Mean±SE) 37.67 ± 2.98 21.10 ± 0.79 1.04 (1.03–1.05) ≤ 0.01 1.03 (1.01–1.04) b 0.01

b 12.15 13 (7.65) 134 (33.33) 1.00&

1.00&

12.15-22.10 36 (21.18) 134 (33.33) 2.77 (1.41–5.46) ≤ 0.01 2.80 (1.26–6.21) 0.01

≥ 22.10 121 (71.18) 134 (33.33) 9.31 (5.01–17.31) ≤ 0.01 6.71 (3.14–14.35) b 0.01

InAs% (Mean ± SE) 7.18 ± 0.58 6.94 ± 0.48 1.00 (0.98–1.02) 0.77 1.00 (0.98–1.03) 0.72

b 2.86 44 (25.88) 134 (33.33) 1.00&

1.00

2.86-6.03 52 (30.59) 134 (33.33) 1.18 (0.74–1.89) 0.48 1.61 (0.91–2.84) 0.10

≥ 6.03 74 (43.53) 134 (33.33) 1.68 (1.08–2.62) 0.02 1.15 (0.66–2.00) 0.62

MMA% (Mean ± SE) 13.19 ± 0.99 7.53 ± 0.36 1.07 (1.05–1.09) ≤ 0.01 1.08 (1.05–1.12) ≤ 0.01

b 3.29 33 (19.41) 134 (33.33) 1.00& _1.00&

3.29-9.08 35 (20.59) 134 (33.33) 1.06 (0.62–1.81) 0.83 1.67 (0.86–3.23) 0.13

≥ 9.08 102 (60.00) 134 (33.33) 3.09 (1.95–4.90) ≤ 0.01 5.56 (3.09–10.00) b 0.01

DMA% (Mean ± SE) 79.63 ± 1.14 85.52 ± 0.58 0.97 (0.95–0.98) ≤ 0.01 0.97 (0.96–0.99) b 0.01

≥ 91.46 34 (20.00) 134 (33.33) 1.00&

1.00&

83.23-91.46 47 (27.65) 134 (33.33) 1.38 (0.84–2.28) 0.21 1.91 (1.03–3.54) 0.04

b 83.23 89 (52.35) 134 (33.33) 2.62 (1.65–4.16) ≤ 0.01 2.60 (1.49–4.55) b 0.01

8-OHdG (ng/mg creatinine) (Mean ± SE) 7.48 ± 0.97 5.95 ± 0.21 2.00 (1.08–3.68) 0.03 1.71 (0.80–3.63) 0.17

b 3.81 56 (32.94) 134 (33.33) 1.00 1.00 3.81-6.52 51 (30.00) 134 (33.33) 0.91 (0.58–1.43) 0.68 1.22 (0.71–2.11) 0.47 ≥ 6.52 63 (37.06) 134 (33.33) 1.13 (0.73–1.73) 0.59 1.00 (0.57–1.73) 0.99 Retinol (μg/mL) (Mean±SE) 1.10 ± 0.04 1.09 ± 0.02 1.06 (0.72–1.55) 0.77 1.00 (0.63–1.58) 0.99 b 0.87 64 (37.65) 139 (34.58) 1.00 1.00 0.87-1.15 50 (29.41) 132 (32.84) 0.82 (0.53–1.28) 0.38 0.90 (0.52–1.54) 0.70 ≥ 1.15 56 (32.94) 131 (32.59) 0.93 (0.60–1.43) 0.74 0.97 (0.58–1.64) 0.91

Alpha-tocopherol (μg/mL) (Mean ±SE) 10.35 ± 0.44 12.42 ± 0.28 0.92 (0.89–0.96) ≤ 0.01 0.94 (0.90–0.98) 0.01

b 9.47 89 (52.35) 139 (34.58) 1.00& 1.00& 9.47-13.60 52 (30.59) 132 (32.84) 0.62 (0.41–0.93) 0.02 0.73 (0.44–1.22) 0.23 ≥ 13.60 29 (17.06) 131 (32.59) 0.35 (0.21–0.56) ≤ 0.01 0.48 (0.27–0.84) 0.01 Lycopene (μg/dL) (Mean±SE) 9.02 ± 0.82 11.19 ± 0.60 0.98 (0.97–1.00) 0.05 1.00 (0.98–1.02) 0.82 b 4.24 74 (43.53) 139 (34.58) 1.00& 1.00 4.24-12.46 59 (34.71) 132 (32.84) 0.84 (0.55–1.27) 0.41 1.07 (0.64–1.78) 0.80 ≥ 12.46 37 (21.76) 131 (32.59) 0.53 (0.34–0.84) 0.01 0.93 (0.53–1.64) 0.80 Beta-carotene (μg/dL) (Mean±SE) 24.68 ± 2.22 22.17 ± 1.16 1.00 (1.00–1.01) 0.28 1.01 (1.00–1.02) 0.06 b 9.49 51 (30.00) 139 (34.58) 1.00 1.00 9.49-23.94 58 (34.12) 132 (32.84) 1.20 (0.77–1.87) 0.43 1.30 (0.75–2.28) 0.35 ≥ 23.94 61 (35.88) 131 (32.59) 1.27 (0.82–1.97) 0.29 1.49 (0.86–2.57) 0.15

SE: standard error.

&

pb0.01 for trend test. Cutoff points for urinary arsenic proﬁle, 8-OHdG and plasma micronutrients were the respective tertiles of the controls.

a

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were found to be significantly positively associated with the OR of UC in a dose–response relationship regardless of unadjustment or adjustment for covariates (Table 2). There was a signi_{ficant dose–} response relationship between plasma lycopene and UC risk under unadjusted analysis. However, this significant association did not exist when we adjusted for other confounders. Urinary 8-OHdG was significantly different between UC cases and controls, but it was not related to the UC odds ratio for the trend test, regardless of unadjust-ment or adjustunadjust-ment for covariates.

A multivariate linear regression was used to explore the potential factors affecting plasma micronutrients in the control group (Table 3). For the urinary arsenic profile, plasma retinol and alpha-tocopherol were significantly negatively correlated with InAs% (p=0.02 and 0.01, respectively). Alpha-tocopherol significantly increased with increasing DMA% (p = 0.05). Thisfinding suggests that the preferred arsenic methylation pro_{file is related to higher alpha-tocopherol.} Further, the levels of alpha-tocopherol and beta-carotene were significantly increased with increasing age. For males, the levels of alpha-tocopherol and beta-carotene were signi_{ficantly lower than} those for females. For lifestyle risk factors, the levels of lycopene and beta-carotene were significantly decreased in people who had higher cumulative cigarette smoking. These results indicated that the elderly, females and non-smokers have higher antioxidants than younger people, males and smokers. No associations between the habits of alcohol drinking or pesticide use and plasma micronutrients were observed in our results.

Further analyses were carried out to assess the joint effects of each plasma micronutrient and total arsenic levels on the urinary 8-OHdG levels in the control group (Fig. 1). Trend analysis revealed progressively increased 8-OHdG through exposure to no risk factor (low total arsenic and high lycopene or low total arsenic and high beta carotene), either one of the factors, or both of the two risk factors (p values for trends were 0.01 and 0.02, respectively) after adjustment for age, gender education, paternal ethnicity, cigarette smoking, alcohol drinking, and pesticide use in multivariate linear regression.

The joint effect of the arsenic methylation profile and plasma alpha-tocopherol on UC risk are shown inTable 4. The significantly increased UC risk was most pronounced in participants who had high total arsenic and low plasma alpha-tocopherol (p≤0.01 for trend test). However, this interaction was statistically significant only in the multiplicative model, but not in the synergy index. The same patterns were shown in the joint effect of InAs%, MMA%, or DMA%, and plasma

alpha-tocopherol on UC risk. However, all synergy index and multi-plicative models were insigniﬁcant.

4. Discussion

To our knowledge, this is the ﬁrst study to evaluate whether plasma alpha-tocopherols could modify UC risk related with poor arsenic methylation in a population without obvious exposure to arsenic. Among these lipophilic antioxidants, we only found the protective predictor of plasma alpha-tocopherols on UC risk. Then, plasma alpha-tocopherol levels were inversely associated with total arsenic and InAs%, and positively associated with DMA% in a multi-variate linear regression model. Lower plasma alpha-tocopherol might modify the risk of inorganic arsenic-related UC.

Since 2000, the allowable arsenic levels in drinking water have decreased from 50 to 10μg/L in Taiwan. According to the Taipei Water Department of the Taipei City Government, the average arsenic concentration in Taipei tap water is 0.7μg/L (range: non-detectable to 4.0μg/L). In the early 1910s, there were two endemic areas of long-term exposure to arsenic from drinking water in Taiwan, including southwestern and northeastern areas. Until the mid-1970s, a public water supply system was generally utilized and artesian well water was no longer used in daily life (Chen et al., 2002). Distinct from previously exposed areas, the majority of our participants (≥80%) lived in Taipei City. Compared to the arsenic-contaminated south-western area in Taiwan (Hsueh et al., 1997), the sums of urinary iAs3±, iAs5±_{, MMA}5±_{and DMA}5±_{of study participants in the present study}

were low (mean value of total arsenic: approximately 70μg/L vs. 25μg/L, respectively). We randomly collected drinking water from 37 UC cases and measured the total arsenic levels; the mean ± standard error was 17.14 ± 0.55μg/L greater than the standard level. In addition to drinking water, arsenic species were recently detected in agricultural rice paddy soils in the Guandu Plain of northern Taiwan (Chiang et al., 2010). Although we do not know the exact exposure source, we still observed a signiﬁcantly increased risk of UC in those with an unfavorable urinary arsenic proﬁle, including higher InAs%, higher MMA% or lower DMA% (Pu et al., 2007).

The standard concentration of arsenic in drinking water and the risk of low doses of arsenic have been discussed throughout the world (Schoen et al., 2004; Snow et al., 2005). Arsenic-induced cytotoxicity caused by high arsenic exposure and mediated through reactive oxygen species was fully evidenced in in-vitro and human studies (Shi

Table 3

Multivariate analysis of the risk factors for plasma micronutrients in the control group.

Variables Retinol (μg/mL) α-tocopherol (μg/mL) Lycopene (μg/dL) β-carotene (μg/dL)

β (SE) p β (SE) p β (SE) p β (SE) p

Age 0.0005 (0.001) 0.49 0.003 (0.001) b0.01 0.001 (0.002) 0.70 0.004 (0.001) 0.01

Gender (male vs. female) 0.04 (0.02) 0.06 −0.06 (0.02) 0.02 0.05 (0.07) 0.47 −0.13 (0.05) 0.01

Education

Elementary school or below

High school −0.02 (0.02) 0.42 0.04 (0.03) 0.14 −0.04 (0.07) 0.57 0.03 (0.05) 0.59

College or above −0.03 (0.03) 0.21 0.07 (0.03) 0.01 0.02 (0.07) 0.78 0.04 (0.06) 0.45

Cumulative cigarette smoking 0.01 (0.02) 0.71 −0.01 (0.02) 0.56 −0.19 (0.06) b0.01 −0.11 (0.05) 0.02

Alcohol drinking Never

Occasional −0.02 (0.02) 0.37 −0.01 (0.02) 0.71 0.05 (0.06) 0.36 0.02 (0.05) 0.66

Regular 0.02 (0.03) 0.45 −0.02 (0.03) 0.53 −0.04 (0.08) 0.62 −0.09 (0.06) 0.12

Pesticide usage (yes vs. no) −0.005 (0.03) 0.89 −0.04 (0.04) 0.30 −0.15 (0.10) 0.12 0.01 (0.07) 0.85

Total arsenic levels 0.002 (0.03) 0.94 −0.06 (0.03) 0.05 −0.04 (0.08) 0.65 0.10 (0.06) 0.08

InAs% −0.05 (0.02) 0.02 −0.07 (0.02) 0.01 −0.10 (0.06) 0.10 −0.09 (0.05) 0.07

MMA% −0.04 (0.02) 0.07 −0.03 (0.02) 0.20 −0.04 (0.07) 0.54 −0.02 (0.05) 0.69

DMA% 0.16 (0.12) 0.17 0.24 (0.12) 0.05 0.15 (0.33) 0.64 0.05 (0.25) 0.83

8-OHdG −0.03 (0.03) 0.32 0.005 (0.03) 0.88 −0.02 (0.09) 0.79 0.07 (0.07) 0.28

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et al., 2004; Basu et al., 2004;Kubota et al., 2006).Moore et al. (1997) demonstrated that chromosome damage in the form of micronuclei in human exfoliated bladder cells are induced by high arsenic exposure. The effects of low dose arsenic exposure should be further evaluated. Our previous study found a signiﬁcantly positive association between urinary arsenic proﬁles (high total arsenic, high iAs3+_{%, high MMA%,}

and low DMA%) and UC risk (Pu et al., 2007); we also found a signiﬁcant increase in 8-OHdG levels related to urinary arsenic species (total arsenic, iAs3+_{, MMA, and DMA) in low arsenic exposure areas}

(Chung et al., 2008). However, only a weak association between urinary 8-OHdG levels and arsenic-associated UC risk was observed.

In addition, one study demonstrated that low levels of arsenic provoked a cellular adaptive oxidative stress response through increasing antioxidant levels (Fu et al., 2010). Therefore, we further evaluated the joint effect of urinary arsenic profile and plasma micronutrient levels on UC risk in the present study. Our study showed that participants who had lower total arsenic and higher lycopene (or beta-carotene) had lower urinary 8-OHdG levels than those with higher total arsenic and lower lycopene (or beta-carotene) (pb0.01 for trend test) in the control group. However, we did not find any association between plasma lipophilic antioxidants and urinary 8-OHdG level in multivariate linear regression models, which is consistent with thefindings ofKimura et al. (2006).

Alpha-tocopherol present in the cell membrane could disrupt lipid peroxyl radical chains, thus preventing further lipid peroxidation (Kadirvel et al., 2007). In addition, alpha-tocopherol was interrelated with reduced glutathione (GSH); ascorbic acid could improve cellular thiol status as well as maintain DNA and proteins in native form through the recycling processes (Meister, 1994b). During the process, tocopheroxyl radicals are transformed by accepting free radicals and then reduced to alpha-tocopherol under reaction with ascorbic acid. Sequentially, the dehydroascorbic acid formed in this reaction is reduced to ascorbic acid with the oxidation of reduced GSH (Meister, 1994a; Brigelius-Flohe and Traber, 1999). Therefore, the antioxidant protection of alpha-tocopherol might be a result of neutralizing ROS directly or regulating GSH recycling indirectly or another enzymatic mechanism (Ramanathan et al., 2002). Exposure to inorganic arsenic in drinking water or further arsenic methylation in the human body might be involved in ROS production (Lynn et al., 2000;Nishikawa et al., 2002;Pi et al., 2002;Wei et al., 2005). In animal models, the administration of alpha-tocopherol and ascorbic acid to arsenic-exposed rats signiﬁcantly reduced oxidative stress and produced cell apoptosis by directly eliminating free radicals (Lynn et al., 2000; Ramanathan et al., 2005;Kadirvel et al., 2007). In our data, 12.42 ± 0.28μg/mL of alpha-tocopherol in healthy controls was similar to 14.3 ± 0.09μg/mL reported by Badjatia et al., and was lower than 27.48 ± 12.92μg/mL reported by Liang et al.; these studies also reported the same signiﬁcant protection on UC risk (Liang et al., 2008; Badjatia et al., 2010). Alpha-tocopherol affects the expression and Fig. 1. The joint effect of plasma micronutrients and total arsenic levels on the urinary

8-OHdG levels in the control group. ⁎ p≤0.05 for multiple linear regression.

Table 4

Ad justed odds ratios and 95% conﬁdence intervals (CI) for the interaction of urinary total arsenic and plasma micronutrients on UC risk. Plasma alpha-tocopherol

(μg/mL)

Urinary arsenic species Cases/controls ORa

(95% CI) Synergy index

(95% CI) pb ≥11.21 Total arsenicb16.60 5/100 1.00 0.97 (0.55–1.69) 0.05 ≥11.21 Total arsenic≥16.60 25/101 5.17(1.74–15.38) b11.21 Total arsenicb16.60 42/97 6.85(2.407–19.55) b11.21 Total arsenic≥16.60 98/104 10.70(3.89–29.47) Ptrendb0.01 ≥11.21 InAs%b4.32 26/108 1.00 1.49 (0.22–10.20) 0.55 ≥11.21 InAs%≥4.32 47/93 1.86 (0.96–3.59) b11.21 InAs%≤ 4.32 21/89 0.77 (0.37–1.66) b11.21 InAs%N4.32 76/112 1.94 (1.03–3.68) Ptrend–0.18 ≥11.21 MMA%b6.10 14/104 1.00 1.42 (0.75–2.70) 0.54 ≥11.21 MMA%≥6.10 32/97 2.65 (1.15–6.11) b11.21 MMA%≤ 6.10 33/93 5.28 (2.29–12.19) b11.21 MMA%N6.10 91/108 9.43 (4.29–20.74) Ptrendb0.01 ≥11.21 DMA%≥88 16/101 1.00 2.29 (0.91–5.74) 0.55 ≥11.21 DMA%b88 26/100 1.66 (0.74–3.71) b11.21 DMA%≥88 31/96 2.47 (1.14–5.38) b11.21 DMA%b88 97/105 5.88 (2.82–12.23) Ptrendb0.01

Total arsenic (μg/g creatinine).

Cutoff points for urinary arsenic proﬁle and plasma alpha-tocopherol were the respective medians of the controls.

a

Adjusted by age, gender, education, paternal ethnicity, cigarette smoking, alcohol drinking, pesticide use and urinary creatinine levels.

b

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activity of the immune and inﬂammatory cells by inhibiting the activity of protein kinase C, an important cell-signaling molecule (Sampayo-Reyes and Zakharyan, 2006). Additionally, glutathione is critical to the metabolism of the arsenic methylation, speciﬁcally the initial reduction of iAs5+_{to iAs}3+₍_{Zakharyan et al., 1999}_{). Perhaps,}

alpha-tocopherol diminishes arsenic toxicology through interaction with glutathione to decrease UC risk. The joint effect of alpha-tocopherol and glutathione on UC risk needs further investigation.

Previous studies demonstrated conflicting findings regarding pesticide use and human cancers (Rusiecki et al., 2004; Kang et al., 2008; Koutros et al., 2009; Chrisman et al., 2009). The United States Agricultural Health Study constructed a prospective cohort of 57,311 licensed pesticide applicators in the United States, enrolled between 1993 and 1997, and evaluated the incidence of cancer developed through 2004. For heterocyclic aromatic amine pesticide users, the study found signi_{ficant trends in risk with increasing} lifetime exposure for bladder cancer and colon cancer (Koutros et al., 2009). However, moderate to weak correlations were observed for bladder cancer in pesticide sales in Brazil (Chrisman et al., 2009). The index of pesticide exposure in our study is a simple yes-or-no variable, and we evaluated it as a confounder to adjust in the multivariate logistic model. Future study may need to explore the association between pesticide use and bladder cancer. In addition, the carcinogens in cigarette smoke, including polycyclic aromatic hydrocarbons, aromatic amines, and other chemicals, are proposed to be involved in ROS production and oxidative damage (Pryor, 1997). In our previous work, we found a significant joint effect of cigarette smoking and arsenic methylation capacity on UC risk (Pu et al., 2007); we did not prove the association between cigarette smoking and oxidative stress, including urinary 8-OHdG levels (Chung et al., 2008). However, lycopene and beta-carotene were signi_{ficantly inversely related with cumulative cigarette smoking in} this study.

The present study attempted to clarify the important role of oxidative stress on arsenic-related UC risk based on biochemical levels. We used the indices of general oxidative stress evaluation, including plasma micronutrients and urinary 8-OHdG, which resulted from environmental exposure. In the present study, we could not clarify the roles of plasma micronutrients on the association between urinary 8-OHdG and UC risk. The role of ROS-related DNA repair enzymes in arsenic carcinogenesis needs further exploration. The accuracy of one-spot evaluation of plasma antioxidants may be in doubt. However, the values might be reliable if all subjects reported no accompanying changes in lifestyle. In addition, the specimen collection and measurement of urinary arsenic and plasma micronutrients were completed at the same time in this study, but they may be an effect modifier of each other on UC risk. Plasma samples were collected since cancer diagnosis, which might underestimate the association between plasma micronutrients and UC risk if UC patients improved their dietary habits after diagnosis. However, we still observed a significant inverse relationship between alpha-tocopherol and UC risk. The information of food frequency in Chinese dietary was not precise and recall bias should be also considered. Therefore, we used the concentrations of plasma micronutrients as a better predictor to evaluate individual nutrition status. We have no information about medication that may affect plasma micronutrient levels, which was a study limitation. Because of the small sample size, statistical significance should be interpreted with caution. Prevalent UC cases were recruited in this study; however, we cannot exclude thefindings about the association between plasma micronutrients or arsenic and its various metabolites and UC, and the fact that these associations might be the result of and not the cause for UC. In conclusion, our results might suggest a potential protective effect of alpha-tocopherol on UC, particularly in study participants with inefficient arsenic methylation capability.

Acknowledgments

The study was supported by grants from the National Science Council of the ROC (NSC 86-2314-B-038-038, NSC 87-2314-B-038-029, NSC-88-2314-B-038-112, NSC-89-2314-B038-049, SC-89-2320-B038-013, NSC-90-2320-B-038-021, NSC 91-3112-B-038-0019, NSC 92-3112-B-038-001, NSC 93-3112-B-038-001, NSC 94-2314-B-038-023, NSC-95-2314-B-038-007, NSC-96-2314-B038-003 and NSC 97-2314-B-038-015-MY3). References

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