Urinary arsenic species and CKD in a Taiwanese population: a case-control study.

Case-Control Study

Yu-Mei Hsueh, PhD,

_{Chi-Jung Chung, MSc,}

_{Horng-Sheng Shiue, MD,}

_{Jin-Bor Chen, MD,}

Shou-Shan Chiang, MD,

_{Mo-Hsiung Yang, PhD,}

_{Cheng-Wei Tai, MSc,}

_{and Chien-Tien Su, MD}

Background: Inorganic arsenic has been linked to decreased kidney function through oxidative

damage. Arsenic methylation is believed to be a pathway for arsenic metabolism. Lycopene is an antioxidant that reduces oxidative stress; however, the association between urinary arsenic species, plasma lycopene level, and chronic kidney disease (CKD) has seldom been evaluated.

Study Design: Case-control study.

Setting & Participants: 125 patients with CKD and 229 controls were recruited from a hospital-based

pool.

Predictor: Urinary arsenic species and plasma lycopene level.

Outcomes & Measurements: CKD was defined as estimated glomerular filtration rate (eGFR) less

than 60 mL/min/1.73 m2_{, calculated by using the Modification of Diet in Renal Disease Study equation.} Plasma lycopene was measured by means of high-performance liquid chromatography. Urinary arsenic species, including arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid, were deter-mined by means of high-performance liquid chromatography and hydride generator–atomic absorption spectrometry.

Results: Lycopene level was associated positively with eGFR, and participants with a high serum

lycopene level had a significant, inverse association with CKD (odds ratio, 0.41; 95% confidence interval, 0.21 to 0.81). Total arsenic level was associated significantly with CKD in a dose-response relationship, especially in participants with a total arsenic level greater than 20.74 compared with 11.78 ␮g/g creatinine or less (odds ratio, 4.34; 95% confidence interval, 1.94 to 9.69). Furthermore, participants with a high urinary total arsenic level or participants with a low percentage of dimethylarsinic acid had a positive association with CKD when their plasma lycopene level was low.

Limitations: Because of the single spot evaluation of plasma antioxidants and urinary arsenic

species and the small sample size, statistical significance should be interpreted with caution.

Conclusions: This study shows that high urinary total arsenic or low plasma lycopene level is

associated positively with CKD. Results suggest that the capacity for arsenic methylation may be associated with CKD in individuals who ingest low arsenic levels in drinking water and also have a low plasma lycopene level.

Am J Kidney Dis 54:859-870. © 2009 by the National Kidney Foundation, Inc.

INDEX WORDS: Arsenic; arsenic methylation capacity; lycopene; chronic kidney disease.

C

hronic kidney disease (CKD) now is

recog-nized as a common condition that

in-creases the risk of cardiovascular disease.

_The

national prevalence of CKD in Taiwanese

pa-tients with an estimated glomerular filtration rate

(eGFR) less than 60 mL/min/1.73 m

_{is 11.93%,}

but only 3.54% of participants are aware of their

disorder.

_{CKD is an important public issue}

because Taiwan ranks first in the world in the

incidence of end-stage renal disease.

Epidemio-logical and clinical evidence have shown a link

between hypertension, diabetes, obesity, and

met-abolic syndrome and the onset and progression

of CKD.

The metalloid arsenic is a naturally occurring

element in soil, food, and water. Humans are

exposed to inorganic arsenic from mining and

smelting metal ores, pesticide manufacturing,

From the1

Department of Public Health, School of Medi-cine; 2

School of Public Health; 3

Department of Chinese Medicine, Chang Gung Memorial Hospital, and Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei;4

Nephrology Division, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung;5

Depart-ment of Internal Medicine/Nephrology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei; 6

Department of Nuclear Sci-ence, National Tsing-Hua University, Hsinchu; and7

Depart-ment of Family Medicine, Taipei Medical University

Hospi-tal, Taipei, Taiwan.

Received December 17, 2008. Accepted in revised form June 5, 2009. Originally published online asdoi: 10.1053/ j.ajkd.2009.06.016on August 17, 2009.

Address correspondence to Yu-Mei Hsueh, PhD, Depart-ment of Public Health, School of Medicine, Taipei Medical University, No. 250 Wu-Hsing St, Taipei 110, Taiwan. E-mail:

ymhsueh@tmu.edu.tw

© 2009 by the National Kidney Foundation, Inc.

0272-6386/09/5405-0011$36.00/0 doi:10.1053/j.ajkd.2009.06.016

wood preservatives, and medicines. Food may

contain both organic and inorganic arsenic,

whereas drinking water contains primarily

inor-ganic arsenic. Long-term exposure to inorinor-ganic

arsenic has been related to risk of cancer in the

skin, bladder, liver, kidney, and lung.

Histori-cally, the arsenic concentration permitted in

pub-lic water supplies in Taiwan was 50

␮g/L.

How-ever, in 2000, a new standard of 10

␮g/L was

announced. Our recent study showed that

indi-viduals with an unfavorable urinary arsenic

pro-file had increased risk of urothelial carcinoma,

even at low levels of exposure.

We do not know

whether a urinary arsenic profile within a low

allowable range affects the risk of CKD.

In comparison to other metals, such as lead

and cadmium, studies of arsenic-induced

nephro-toxicity are rare. However, a report is available

for arsenic-induced kidney damage.

A recent

study from Michigan also has shown an

in-creased rate of kidney disease in people exposed

to arsenic-contaminated drinking water.

The

mechanisms underlying arsenic-induced kidney

toxicity are complex.

Absorbed arsenic undergoes complicated

biomethylation to form monomethylarsonic

acid (MMA

[the superscript indicates an

oxi-dation number of 5 for arsenic]) and

dimethyl-arsinic acid (DMA

), which are excreted by

the kidneys into urine.

The presumed arsenic

methylation pathway in the human body is

shown in

Fig 1

.

A previously published

report suggests that excessive generation of

reactive oxygen species (ROS) by various

met-als may cause kidney damage.

Because

ar-senic also generates ROS during the metabolic

activation process,

whether arsenic

metabo-lites are part of the mechanism for

arsenic-induced nephrotoxicity remains to be

deter-mined.

Lycopene is a potent carotenoid antioxidant.

Lycopene most likely is involved in the

scaveng-ing ROS that contribute to defense against lipid

peroxidation.

A recent study has shown that

lycopene is able to protect against mercuric

chlo-ride–induced nephrotoxicity in rats,

as well as

cisplastin-induced decreased kidney function and

oxidative stress in rats.

_{Low plasma lycopene}

levels and arsenic exposure may be a risk factor

for CKD. Therefore, the primary goal of the

present study is to examine the association

be-tween the capacity for arsenic methylation,

lyco-pene level, and CKD and the interaction between

the capacity for arsenic methylation and

lyco-pene level in affecting CKD.

METHODS

Study Participants, Interview

Process, and Measurements

On a weekly basis from September 2005 and December 2007, patients (age range, 22 to 88 years) with clinical evidence of CKD based on urine sample collection were recruited from the Department of Internal Medicine/Nephrol-ogy of Shin Kong Wu Ho-Su Memorial Hospital in Taipei, Taiwan, resulting in 125 participants. eGFR traditionally is considered the best overall index of kidney function in health and disease. We used the 4-variable equation from the Modification of Diet in Renal Disease (MDRD) Study1_to

estimate eGFR as 186.3 ⫻ (serum creatinine)⫺1.154 ⫻ (age)⫺0.203⫻ (0.742 for female) and defined the 5 stages of CKD according to the relevant Kidney Disease Outcomes Quality Initiative guidelines from the National Kidney Foun-dation. In this study, participants who were in stages 3 to 5 (eGFR⬍ 60 mL/min/1.73 m2_{) for 3 months were defined as}

having CKD. Age frequency–matched control participants with no evidence of CKD (eGFRⱖ 60 mL/min/1.73 m2_{) in a}

2:1 ratio of controls to cases were accrued weekly from a hospital-based pool, including those receiving senior citizen health examinations at Taipei Medical University Hospital and those receiving adult health examinations at Taipei Municipal Wan Fang Hospital. A total of 229 control partici-pants was obtained, and a urine sample was collected from each.

Well-trained personnel carried out standardized personal interviews based on a structured questionnaire. The informa-tion collected included demographic and socioeconomic characteristics and potential risk factors for CKD, such as lifestyle, alcohol consumption, cigarette smoking, exposure to potential occupational and environmental carcinogens (hair dyes and pesticides), medication history, consumption of conventional and alternative medicines, and personal and family histories of hypertension, diabetes, and CKD.

Figure 1. The presumed arsenic methylation pathway in the human body. The numbered steps are catalyzed by the following enzymes: (1) arsenate reductase or purine nucleoside phosphorylase (PNP), (2) arsenite methyl trans-ferase (As3MT), (3) glutathione S-transtrans-ferase omega 1 or 2 (GSTO1, GSTO2), and (4) arsenite methyl transferase (As3MT). Abbreviations: DMA, dimethylarsinic acid; MMA, monomethylarsonic acid; SAHC, S-adenosylhomocys-teine; SAM, S-adenosylmethionine.

The Research Ethics Committee of Taipei Medical Univer-sity (Taipei, Taiwan) approved the study. All patients pro-vided informed consent forms before sample and data collec-tion. The study was consistent with the World Medical Association Declaration of Helsinki.

A 10-mL blood sample was collected from participants on recruitment by use of EDTA-treated vacuum syringes and disposable needles. Plasma samples were centrifuged at 3,000 rpm for 15 minutes at room temperature, separated into aliquots, and stored at⫺80°C until used. Spot urine samples also were collected from all participants and imme-diately transferred to a⫺20°C freezer until further use for urinary arsenic species analysis.

Determination of Urinary Arsenic Species

It has been shown that urinary arsenic species are stable for at least 6 months when preserved at⫺20°C.21_Therefore,

the urine assay was performed within 6 months after collec-tion. Frozen urine samples were thawed at room tempera-ture, dispersed by using ultrasonication, filtered through a Sep-Pak C18column (Mallinckrodt Baker Inc, Phillipsburg,

NJ) and levels of arsenite (As[III]), arsenate (As[V]), MMAV_,

and DMAV_{were determined. A urine aliquot of 200␮L was}

used for determination of arsenic species by using high-performance liquid chromatography (HPLC; Waters 501; Waters Associates, Milford, MA) with columns obtained from Phenomenex (Nucleosil, Torrance, CA). Inorganic ar-senic and its metabolites were quantified by using hydride generator–atomic absorption spectrometry.22_{A standard}

so-lution of 4 arsenic species was prepared in our laboratory; the sample and sample-spiked standard solution were deter-mined by using online HPLC–hydride generator–atomic absorption spectrometry. Recovery rates of the 4 arsenic species were calculated by using the following formula: [(sample-spiked standard solution concentration⫺ sample concentration)/(standard solution concentration)] ⫻ 100. Recovery rates for As(III), DMAV_{, MMA}V_{, and As(V)}

ranged between 93.8% and 102.2%, with detection limits of 0.02, 0.06, 0.07, and 0.10␮g/L, respectively. The urinary concentration of the sum of inorganic arsenic, MMAV_{, and}

DMAV _{was normalized against urinary creatinine levels}

(micrograms per gram of creatinine). The colorimetric assay automatically determined by the Roche Modular P800 instru-ment (Roche Inc, Mannheim, Germany) was used to calcu-late creatinine level by measuring the creatinine–picric acid complex formed by the reaction of creatinine and picric acid. The standard reference material, SRM 2670, contains 480⫾ 100␮g/L of inorganic arsenic and was obtained from the National Institute of Standards and Technology (Gaithers-burg, MD). SRM 2670 was used as a quality standard and analyzed along with urine samples. The mean value of SRM 2670 determined by our system was 507⫾ 17␮g/L (n ⫽ 4). The arsenic methylation indices were assessed by the percent-ages of various urinary arsenic species present in the sum of inorganic arsenic, MMAV_{, and DMA}V_{. The primary}

methyl-ation index was defined as the ratio of MMAV_{to levels of}

inorganic arsenic, ie, As(III)⫹ As(V), and the secondary methylation index was defined as the ratio of DMAV _to

MMAV_.23

Determination of Plasma Antioxidant

Micronutrient Level

Levels of␤-carotene, lycopene, ␣-tocopherol, and retinol in plasma samples were measured by using HPLC according to the procedure described previously.24_{Analysis was}

car-ried out by using reversed-phase HPLC (Hitachi Inc, Tokyo, Japan) with a mobile phase consisting of methanol:acetoni-trite:chloroform (47:47:6) and multiwave length monitoring. Retinol was detected at 325 nm;␣-tocopherol, at 280 nm; and lycopene and␤-carotene, at 466 nm. Plasma samples for each case and control set were thawed from⫺80°C in dim light at room temperature and assayed on the same day to ensure that temporal variability in laboratory assays would affect cases and controls equally. All laboratory personnel were unaware of the disease status of participants from whom plasma samples were tested. Recovery rates for ␤-carotene, lycopene, ␣-tocopherol, and retinol were 90% to 100% at the highest concentration and 90% to 107% at the lowest concentration of the standard solution. The precision (coefficient of variance) of␤-carotene, lycopene, ␣-tocoph-erol, and retinol was 1.0% to 6.0%. We also used an internal control (␣-tocopherol acetate) to reduce systematic error; the coefficient of variance for␣-tocopherol acetate was 2.5%.

Statistical Analysis

Continuous variables are expressed as mean⫾ SE. Stu-dent t test was used to compare differences in urinary arsenic profiles between case participants and controls. Analysis of variance and Scheffe multiple comparison correction were applied to compare urinary arsenic profiles between the varied exposure strata. Unconditional logistic regression models were used to estimate multivariate-adjusted odds ratio (OR) and 95% confidence interval (CI). Cutoff values for continuous variables were the respective tertiles of controls. Significance tests for linear trend among ORs across exposure strata were calculated by categorizing expo-sure variables and treating scored variables as continuous. For joint-effect analysis, cutoff values for plasma lycopene, urinary arsenic species percentage, or arsenic methylation indices were the respective medians of the controls. The synergy index proposed by Rothman25 _{was computed to}

assess the additive interaction relationship between lyco-pene levels and urinary arsenic species percentages or ar-senic methylation indices on CKD risk. An observed syn-ergy index value that departs substantially from the expected additive null, ie, a synergy index not equal to 1, suggests an additive interaction effect. ORs and variance covariance matrixes then were used to calculate values for synergy index and 95% CIs.26

RESULTS

Participants who had higher educational levels

had a significantly lower risk of CKD than those

with lower educational levels. Participants with

diabetes or hypertension had a significantly greater

CKD risk than those without diabetes (OR, 4.00;

95% CI, 2.04 to 7.76) or those with normal blood

pressure (OR, 2.23; 95% CI, 1.34 to 3.70).

Alco-hol consumption was related to a significantly lower

CKD risk than for nondrinkers. Cigarette smoking

was not associated with CKD risk. A significantly

greater risk was shown in analgesic users than

nonusers; however, analgesic use on an as-needed

basis had a significantly lower CKD risk than in

nonusers (

Table 1

). Coffee consumption, pesticide

exposure, and paint or dye use did not affect risk

of CKD (data not shown).

The CKD group had a significantly lower

eGFR (28.40

⫾ 1.41 mL/min/1.73 m

; n

⫽ 125)

than controls (80.17

⫾ 1.21 mL/min/1.73 m

;

n

⫽ 229; P ⬍ 0.001;

Fig 2

). Patients with CKD

had a significantly greater urinary total arsenic

level, greater MMA

percentage, lower DMA

percentage, and lower plasma lycopene level

than controls (

Table 2

).

Plasma lycopene level was positively

associ-ated and urinary total arsenic level was

nega-tively associated with eGFR (both associations

were statistically significant;

Fig 3

), whether

adjusted for age and sex or multiple covariates.

When eGFR was adjusted for multiple

covari-ates, greater MMA

percentages correlated with

significantly lower eGFRs (ie, inverse

correla-tion), and greater DMA

percentages correlated

with significantly greater eGFRs (data not

shown).

Compared with men, women had lower

MMA

percentages, but significantly greater

to-tal arsenic levels. Cigarette smoking, alcohol

consumption, and habitual analgesic use did not

influence the arsenic profile (

Table 3

).

By performing trend analysis on urinary total

arsenic level, percentage of arsenic species, or

plasma lycopene strata in tertiles, total urinary

arsenic level was associated significantly with the

CKD OR in a dose-response relationship, as listed

in

Table 4

. This was especially true in participants

with a total arsenic level greater than 20.74

␮g/g

creatinine, in whom the OR of CKD was increased

4-fold compared with those with a total arsenic

Variables CKD Group Healthy Controls

Odds Ratio* (95% confidence interval) P Sex Men 59 (47.20) 91 (39.74) 1.00 Women 66 (52.80) 138 (60.26) 0.77 (0.49-1.20)† 0.3 Age (y) 58.81_{⫾ 13.96} 60.61_{⫾ 13.09} 0.99 (0.97-1.00)‡ 0.2 Educational level Illiterate/elementary school 60 (48.00) 63 (27.75) 1.00

Junior/senior high school 40 (32.00) 69 (30.40) 0.45 (0.25-0.78) 0.005

ⱖCollege 25 (20.00) 95 (41.85) 0.13 (0.07-0.27) _⬍0.001 Cigarette smoking No 100 (80.65) 178 (77.73) 1.00 Yes 24 (19.35) 51 (22.27) 0.65 (0.35-1.21) 0.2 Alcohol consumption Never 103 (82.40) 147 (64.19) 1.00 Frequency 12 (9.60) 32 (13.97) 0.37 (0.17-0.79) 0.01 Occasional 10 (8.00) 50 (21.83) 0.20 (0.10-0.44) _⬍0.001 Diabetes No 81 (75.70) 210 (92.11) 1.00 Yes 26 (24.30) 18 (7.89) 4.00 (2.04-7.76) _⬍0.001 Hypertension No 64 (59.81) 174 (76.32) 1.00 Yes 43 (40.19) 54 (23.68) 2.23 (1.34-3.70) 0.002 Analgesic use No 97 (77.60) 173 (75.88) 1.00 Yes, routinely 14 (11.20) 9 (3.95) 3.00 (1.24-7.27) 0.02

Yes, as the need arises 14 (11.20) 46 (20.18) 0.53 (0.28-1.01) 0.05

Note: Values expressed as number (percent) or mean⫾ SE unless noted otherwise.

Abbreviation: CKD, chronic kidney disease. *Adjusted for age and sex, except where indicated. †Adjusted only for age.

level of 11.78

␮g/g creatinine or less. Other arsenic

species indices were not related to the CKD OR.

Plasma lycopene level was related inversely to

CKD in a dose-response relationship (participants

with a plasma lycopene level

⬎ 18.64

␮g/dL

com-pared with

ⱕ 8.29

␮g/dL; OR, 0.41; 95% CI, 0.21

to 0.81). Plasma retinol level was associated

signifi-cantly with CKD risk (data not shown), whereas

other micronutrients were not related to CKD (data

not shown).

Additional analyses were carried out to assess

the joint effects of the following pairs of factors

on CKD risk: lycopene and total arsenic levels,

lycopene level and percentage of arsenic species,

or lycopene level and arsenic methylation

indi-ces (

Fig 4

). Trend analysis showed progressively

Table 2. Differences in Urinary Total Arsenic, Percentages of Arsenic Species, and Arsenic Methylation Indices Between the CKD Group and Healthy Controls

Variables

CKD Group Healthy Controls

P

No. Tested Value No. Tested Value

Total arsenic (␮g/g creatinine) 124 31.95_{⫾ 2.59} 229 20.71_{⫾ 1.10 ⬍0.001}

Arsenic species (%)

Inorganic arsenic 125 7.50_{⫾ 1.04} 229 6.67_{⫾ 0.62} 0.5

DMA 125 82.02_{⫾ 2.05} 229 87.04_{⫾ 0.83} 0.03

MMA 125 10.49_{⫾ 1.77} 229 6.29_{⫾ 0.49} 0.02

Primary methylation index 120 2.97_{⫾ 0.55} 219 2.37_{⫾ 0.42} 0.4

Secondary methylation index 97 29.68_{⫾ 5.58} 164 26.03_{⫾ 3.63} 0.6

Lycopene (␮g/dL) 125 6.22_{⫾ 1.43} 229 10.40_{⫾ 0.89 ⬍0.001}

Note: Values expressed as mean⫾ SE. Total arsenic indicates inorganic arsenic ⫹ MMA ⫹DMA. Abbreviations: CKD, chronic kidney disease; DMA, dimethylarsinic acid; MMA, monomethylarsonic acid.

increased risks through exposure to no risk

factor, 1 risk factor, or both 2 risk factors.

Although plasma lycopene level tended to

in-teract additively with total urinary arsenic level,

percentage of inorganic arsenic, primary

meth-ylation index, and secondary methmeth-ylation

in-dex in modifying CKD risk, the interactions

were all statistically insignificant, as shown by

the absence of a substantial deviation from 1 in

the synergy index. We also assessed the

inter-action as a departure from joint multiplicative

effects by using the product term of 2 risk

factors and showed that total arsenic level and

DMA percentage significantly interacted with

lycopene level (

Fig 4

).

DISCUSSION

The present study showed that patients with

CKD compared with control individuals had a

significantly greater total urinary arsenic level,

greater MMA

_{percentage, and lower DMA}

percentage, indicating a less efficient capacity to

methylate inorganic arsenic to DMA

_{. In}

addi-tion, it was found that only total urinary arsenic

level was related to CKD risk in a dose-response

̌ʳːʳˈ˅ˁ˃˅ˋ̋ʳʾʳˈˆˁ˄˅ˈ ˥˅ʳːʳ˃ˁ˃ˇˉˇʳʻ̃ˏ˃ˁ˃˃˄ʼ ˃ ˅˃ ˇ˃ ˉ˃ ˋ˃ ˄˃˃ ˄˅˃ ˄ˇ˃ ˄ˉ˃ ˄ˋ˃ ˅˃˃ ˃ ˃ˁ˅ ˃ˁˇ ˃ˁˉ ˃ˁˋ ˄ ˄ˁ˅ ̌ʳːʳˀ˃ˁ˅ˋˊˈ̋ʳʾʳˊ˃ˁˇ˄ˉ ˥˅_{ʳːʳ˃ˁ˃ˇˈ˄ʳʻ̃ˏ˃ˁ˃˃˄ʼ} ˃ ˅˃ ˇ˃ ˉ˃ ˋ˃ ˄˃˃ ˄˅˃ ˄ˇ˃ ˄ˉ˃ ˄ˋ˃ ˅˃˃ ˃ ˈ ˅ ˃ ˃ ˅ ˃ ˈ ˄ ˃ ˃ ˄ ˃ ˈ ˃ Lycopene (μg/dL)

Total arsenic ( μg/g creatinine)

eGFR (m L/min/1.73 m 2) eGFR (m L/min/1.73 m 2)

Figure 3. The association between estimated glomerular filtration rate (eGFR) and plasma lycopene or urinary total arsenic level.

relationship adjusted for age and sex or

sepa-rately adjusted for multiple risk factors. Patients

with CKD had significantly lower plasma

lyco-pene levels, indicating lower antioxidant

capabili-ties than controls.

Upon entering the body, arsenic targets

ubiqui-tous enzyme reactions and affects nearly all

organ systems.

Several trace elements,

includ-ing arsenic, cadmium, lead, and mercury, have

been implicated in the decrease in kidney

func-tion.

A study in Utah has shown increased rates

of nephritis and nephrosis in people drinking

arsenic-contaminated well water.

According to

animal studies, vacuolation of renal tubular

epi-thelium was observed in a case of low-dose

arsenic exposure, whereas pathologically

moder-ate glomerular sclerosis and severe tubular

necro-sis were shown in the case of exposure to high

doses of arsenic.

However, a case report by

Prasad and Rossi

showed that tubulointerstitial

nephritis is associated with increased urinary

arsenic concentration.

According to the Taipei Water Department of

the Taipei City Government, average arsenic

concentration in Taipei tap water is 0.7

␮g/L and

ranges from undetectable to 4.0

␮g/L. However,

the concentration range of urinary arsenic of

study participants of approximately 20 to 30

␮g/g creatinine in this study possibly resulted

from exposure to some foods. Although our

study participants drank tap water with no

evi-dence of arsenic contamination, we also found

that total urinary arsenic level and MMA

per-centage were associated significantly with

de-creased eGFR in this study. However, the precise

mechanism of arsenic-induced nephrotoxicity

may be difficult to assess because of the complex

biological chemistry associated with arsenic.

Absorbed arsenic is excreted mainly through

urine, suggesting that the kidney is a primary

target for arsenic toxicity. Kidney arsenic

toxic-ity may be complicated by methylation of

inor-ganic arsenic to the less toxic MMA

and DMA

,

which are excreted rapidly by the kidney.

MMA

and DMA

have been identified in

human urine.

Many studies have shown that

these trivalent methylated arsenic species are

more toxic than inorganic compounds.

According to Sex, Cigarette Smoking, Alcohol Consumption and Analgesic Use

Variables No. of Participants Total Arsenic (␮g/g creatinine) Arsenic Species (%) PMI SMI Lycopene (␮g/dL) Inorganic

Arsenic MMA DMA

Sex Men 150 21.72⫾ 1.55 6.50⫾ 0.56 9.22 ⫾ 1.12 84.28 ⫾ 1.32 2.44 ⫾ 0.46 25.63 ⫾ 4.24 13.88⫾ 1.09 Women 204 26.84⫾ 1.71 7.31⫾ 0.85 6.71 ⫾ 0.91 85.99 ⫾ 1.24 2.69 ⫾ 0.47 29.01 ⫾ 4.45 14.83⫾ 0.91 P 0.03 0.4 0.08 0.4 0.7 0.6 0.5 Cigarette smoking No 277 24.79⫾ 1.30 6.89⫾ 0.58 7.27 ⫾ 0.79 85.84 ⫾ 1.00 2.62 ⫾ 0.41 27.35 ⫾ 3.43 15.06⫾ 0.77 Yes 75 24.23⫾ 2.88 7.26⫾ 1.41 9.63 ⫾ 1.59 83.12 ⫾ 2.19 2.45 ⫾ 0.46 27.75 ⫾ 6.95 12.25⫾ 1.64 P 0.9 0.8 0.2 0.2 0.8 0.9 0.1 Alcohol consumption No 249 24.89⫾ 1.93 7.08⫾ 0.65 7.76 ⫾ 0.91 85.16 ⫾ 1.15 2.83 ⫾ 0.45 30.18 ⫾ 3.98 14.75⫾ 0.86*, † Yes 44 23.96⫾ 2.89 5.30⫾ 0.48 7.64 ⫾ 2.06 87.06 ⫾ 2.13 2.09 ⫾ 0.63 30.98 ⫾ 10.60 10.26 ⫾ 1.20 Occasional 60 24.22⫾ 4.50 7.71⫾ 1.73 7.90 ⫾ 0.93 84.39 ⫾ 1.91 1.92 ⫾ 0.26 14.45 ⫾ 1.65 16.13⫾ 1.78 P 0.9 0.5 0.9 0.7 0.5 0.1 0.06 Analgesic use No 269 25.12⫾ 1.45 7.00⫾ 0.56 7.47 ⫾ 0.80 85.53 ⫾ 0.99 2.49 ⫾ 0.38 26.65 ⫾ 3.19 13.66⫾ 0.70 Yes, routinely 23 25.82⫾ 3.74 10.35 ⫾ 4.34 7.64 ⫾ 3.38 82.01 ⫾ 5.66 2.40 ⫾ 0.93 46.12 ⫾ 23.30 19.25 ⫾ 3.84 Yes, as needed 60 22.37⫾ 2.12 5.53⫾ 1.15 9.29 ⫾ 1.73 85.25 ⫾ 2.09 3.11 ⫾ 0.91 24.37 ⫾ 7.18 15.46⫾ 2.15 P 0.8 0.3 0.8 0.8 0.9 0.5 0.2

Note: Values expressed as mean⫾ SE. Total arsenic indicates inorganic arsenic ⫹ MMA ⫹ DMA. Cigarette smoking

history and analgesic use data were unavailable for 1 and 2 participants, respectively.

Abbreviations: CKD, chronic kidney disease; DMA, dimethylarsinic acid; MMA, monomethylarsonic acid; PMI, primary methylation index; SMI, secondary methylation index.

*Significantly different from those who consume alcohol, P⬍ 0.05. †Significantly different from occasional drinker, P⬍ 0.05.

ever, trivalent methylated arsenic metabolites

have a short half-life. Whether they can be

de-tected depends on the conditions and

tempera-ture of sample storage and concentrations in

urine. The reason we did not observe trivalent

methylated metabolites in the study is that the

analytical method used lacks the requisite

speci-ficity. In general, arsenic methylation is

consid-ered a detoxification process in which MMA

and DMA

generally are considered nontoxic.

Few studies have examined arsenic metabolism

on decreased kidney function in humans. One

study reported that the main detectable species

were the relatively nontoxic compounds

arseno-betaine and DMA, whereas levels of such toxic

inorganic arsenic compounds as arsenite and

arsenate were less than the detection limit in

serum.

In the present study, we found that

patients with CKD had significantly greater

uri-nary total arsenic levels, greater MMA

percent-ages, and lower DMA

percentages than

con-trols. Of these variables, only total arsenic level

Species, Arsenic Methylation Indices, and Plasma Lycopene

Variables CKD Group/Healthy Controls Odds Ratio* (95% confidence interval) Odds Ratio† (95% confidence interval)

Total arsenic (␮g/g creatinine) Ptrend⬍ 0.001 Ptrend⬍ 0.001

ⱕ11.78 19/75 1.00 1.00

11.78-20.74 30/78 1.73 (0.89-3.40) 1.41 (0.62-3.19)

⬎20.74 76/76 5.66 (2.96-10.85)‡ 4.34 (1.94-9.69)‡

Arsenic species (%)

Inorganic arsenic Ptrend⫽ 0.8 Ptrend⫽ 0.6

ⱕ2.75 40/75 1.00 1.00

2.75-5.86 40/78 0.86 (0.50-1.51) 1.01 (0.52-1.98)

⬎5.86 45/76 0.99 (0.58-1.72) 1.20 (0.61-2.36)

MMA Ptrend⫽ 0.9 Ptrend⫽ 0.7

ⱕ1.29 39/75 1.00 1.00

1.29-7.60 43/77 1.03 (0.60-1.78) 0.63 (0.32-1.23)

⬎7.60 43/77 0.97 (0.56-1.68) 0.87 (0.45-1.71)

DMA Ptrend⫽ 0.7 Ptrend⫽ 0.5

ⱕ85.62 45/76 1.00 1.00

85.62-93.40 44/77 1.00 (0.59-1.70) 0.58 (0.30-1.13)

⬎93.40 36/76 0.88 (0.50-1.53) 0.79 (0.40-1.55)

PMI Ptrend⫽ 0.6 Ptrend⫽ 0.7

ⱕ0.28 40/83 1.00 1.00

0.28-1.86 42/73 1.12 (0.65-1.93) 0.75 (0.38-1.49)

⬎1.86 43/73 1.14 (0.66-1.96) 0.88 (0.46-1.69)

SMI Ptrend⫽ 0.8 Ptrend⫽ 0.4

ⱕ8.44 61/119 1.00 1.00

8.44-17.18 26/54 0.90 (0.51-1.58) 0.48 (0.24-0.99)§

⬎17.18 38/56 1.31 (0.78-2.20) 0.79 (0.42-1.50)

Plasma lycopene (␮g/dL) Ptrend⬍ 0.001 Ptrend⫽ 0.003

ⱕ8.29 74/76 1.00 1.00

8.29-18.64 24/76 0.31 (0.18-0.55)‡ 0.33 (0.17-0.64)_储

⬎18.64 27/77 0.35 (0.21-0.61)_储 0.41 (0.21-0.81)_储

Note: Total arsenic indicates inorganic arsenic⫹ MMA ⫹ DMA.

Abbreviations: CKD, chronic kidney disease; DMA, dimethylarsinic acid; MMA, monomethylarsonic acid; PMI, primary methylation index; SMI, secondary methylation index.

*Adjusted for age and sex.

†Adjusted for age, sex, educational level, paternal and maternal ethnicity, cigarette smoking, coffee drinking, analgesic use, hypertension, and diabetes history.

‡P⬍ 0.001. §P⬍ 0.05. 储P ⬍ 0.01.

Figure 4. Multiple logistical regression analysis of the combination of urinary total arsenic, arsenic species percentage, and plasma lycopene on chronic kidney disease. * P⬍ 0.05; ** P ⬍ 0.01; *** P ⬍ 0.001. The unit of total arsenic is␮g/g creatinine. The relative proportion of each arsenic species (% inorganic As, % MMA and % DMA) was calculated by dividing the levels of each species by the total arsenic level. Abbreviations: PMI, primary methylation index; SMI, secondary methylation index. High is defined as a value greater than the median; low, as a value equal to the median or less. Odds ratios (ORs) based on analyses adjusted for age, sex, educational level, paternal and maternal ethnicity, cigarette smoking, coffee drinking, hypertension, and diabetes history. P represents statistical interaction as a departure from joint multiplicative effects.

was associated significantly with CKD risk.

Whether the capacity of arsenic methylation is

related to patients with CKD when they ingest

low arsenic levels in drinking water needs

fur-ther investigation.

Inorganic arsenic-induced oxidative damage

results in chronic kidney pathological states

in-volving ROS production,

reduction/oxidation-related gene expression, and cytotoxicity.

How-ever, oxidative stress has been identified as an

important mechanism in arsenic-induced

de-creased kidney function through accumulation of

arsenic in kidney tissue; increased levels of

se-rum urea nitrogen, creatinine, and lipid

peroxida-tion end products; and reduced glutathione in a

mouse model.

Our recent study showed that

arsenic methylation species were associated with

oxidative damage assessed by using urinary

8-hy-droxy-2=-deoxyguanosine,

suggesting that

ar-senic metabolites are related to oxidative stress.

Antioxidants could be considered an

alterna-tive approach to mitigate arsenic-induced

oxida-tive damage.

In our previous study, a

signifi-cant inverse dose-response relationship was

observed between arsenic-related ischemic heart

disease and serum

␣- and ␤-carotene levels.

Our study also showed that serum

␤-carotene

level was related negatively to arsenic-induced

skin cancer.

In the present study, we found that

participants with high plasma lycopene levels

had a significantly decreased risk of CKD

com-pared with patients with low plasma lycopene

levels. Additionally, participants with low plasma

lycopene levels were at greater risk of having

CKD when they presented with at least 1 of high

total arsenic level or low DMA

percentage.

Although these data suggest that participants

with low antioxidant capacity may not easily

mitigate oxidative stress produced by arsenic

metabolites and therefore may be at risk of CKD,

these findings need additional study.

Our study had some important limitations that

need to be considered when interpreting results.

First, there is the possibility of selection bias

because cases and controls were recruited from 2

different hospitals; however, bias was minimized

because these hospitals both belonged to medical

centers and were located in Taipei. Furthermore,

the majority of cases and controls lived in Taipei

and were similar in age and sex distribution

(

Table 1

) with respect to demographic

character-istics. Possible selection bias may have occurred

because the recruited CKD cases more often had

an elementary school education than controls.

However, in a large-scale screening program, it

has been reported that participants with a high

level of education had lower CKD risk than those

with a low level of education in Taiwan.

Sec-ond, the accuracy of a single spot evaluation of

plasma antioxidants and urinary arsenic species

may be in doubt. However, the values might be

reliable because all participants had no change in

lifestyle and appeared to maintain their

homeo-static metabolism. Third, because of the small

sample size, statistical significance should be

interpreted with caution. Fourth, CKD cases were

recruited in this study; however, we cannot

ex-clude that the findings of an association between

lycopene or arsenic and its various metabolites

and CKD might be the result and not the cause of

CKD.

In conclusion, this is the first study showing

that high urinary total arsenic levels or low

plasma lycopene levels are associated positively

with CKD. Similarly, our data suggest that the

capacity for arsenic methylation may be

associ-ated with CKD in individuals who also had low

plasma lycopene levels when they ingested low

arsenic levels in drinking water.

ACKNOWLEDGEMENTS

Support: The study was supported by grants

SKH-TMU-95-23, NSC- 96-2314-B038-003 and NSC 97-2314-B-038-015-MY3 from Shin Kong Wu Ho-Su Memorial Hospital in Taipei, Taiwan and National Science Council of the ROC, Taiwan.

Financial Disclosure: None.

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