Arsenic Exposure Area
Chao-Yuan Huang, Jan-Show Chu, Yeong-Shiau Pu, Hsiu-Yuan Yang, Chia-Chang Wu, Chi-Jung Chung and Yu-Mei Hsueh*
From the Graduate Institute of Clinical Medicine (CYH, JSC), Department of Pathology (JSC), and Department of Public Health, School of Medicine (HYY, CJC, YMH), College of Medicine, Taipei Medical University, and School of Public Health, College of Public Health and Nutrition, Taipei Medical University (CCW, YMH); Department of Urology, Taipei Medical University-Shuang Ho Hospital (CCW), and Department of Urology, National Taiwan University Hospital, College of Medicine, National Taiwan University (CYH, YSP), Taipei, Taiwan
Purpose: We explored the relationship between urinary total arsenic and risk of renal cell carcinoma, and investigated whether having hypertension or a low estimated glomerular filtration rate would modify the risk of renal cell carci-noma.
Materials and Methods: The case-control study was conducted between Novem-ber 2006 and May 2009 with 132 patients with renal cell carcinoma, and 260 sex and age matched controls from a hospital based pool. Pathological verification of renal cell carcinoma was completed by image guided biopsy or surgical resection of renal tumors. Urinary arsenic species, including inorganic arsenic, mono-methylarsonic acid and dimethylarsinic acid, were determined with a high per-formance liquid chromatography linked hydride generator and atomic absorption spectrometry. Estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease Study equation.
Results: Urinary total arsenic was significantly associated with renal cell carci-noma risk in a dose-response relationship after multivariate adjustment. Low estimated glomerular filtration rate or hypertension was significantly related to renal cell carcinoma risk. Estimated glomerular filtration rate was significantly negatively related with urinary total arsenic. A significant interaction was seen between the urinary total arsenic and hypertension on renal cell carcinoma risk. The greatest odds ratio (6.01) was seen in the subjects with hypertension, low estimated glomerular filtration rate and high urinary total arsenic. A trend test indicated that the risk of renal cell carcinoma increased along with the accumu-lating number of these 3 risk factors (p⬍0.0001).
Conclusions: Higher urinary total arsenic level was a strong predictor of renal cell carcinoma, and estimated glomerular filtration rate or hypertension interacts with urinary total arsenic in modifying the risk of renal cell carcinoma.
Key Words: arsenic; methylation; kidney; glomerular filtration rate;
carcinoma, renal cell
RENAL cell carcinoma accounts for
85% of renal cancers and for 3% of all adult malignant neoplasms with a male-to-female ratio of 3:2.1 In
Tai-wan 435 males and 230 females had RCC in 2006, which accounted for 0.9% of all patients with cancer.2 Mul-tiple potential etiologic factors
con-Abbreviations and Acronyms AsIII⫽ arsenite AsV⫽ arsenate
DMAV⫽ dimethylarsinic acid eGFR⫽ estimated glomerular filtration rate
MMAV⫽ monomethylarsonic acid RCC⫽ renal cell carcinoma UC⫽ urothelial carcinoma
Submitted for publication August 25, 2010. Supported by grants from the National Sci-ence Council of the Republic of China (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, 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, NSC-97-2314-B-038-015-MY3,1–3 NSC-97-2314-B-038-015-MY3,2–3 NSC-97-2314-B-038-015-MY33–3).
Study received research ethics committee ap-proval.
Supplementary material for this article can be obtained athttp://web2.tmu.edu.tw/ymhsueh.
* Correspondence: Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, No. 250 Wu-Hsing St., Taipei 110, Taiwan (telephone: 886-2-27361661 ext. 6513; FAX: 886-2-27384831; e-mail: ymhsueh@ tmu.edu.tw).
For another article on a related topic see page2353.
2040 www.jurology.com
0022-5347/11/1856-2040/0 Vol. 185, 2040-2044, June 2011
THE JOURNAL OF UROLOGY® Printed in U.S.A.
tribute to the development of RCC including obesity, cigarette smoking, and exposure to trichloroethyl-ene, asbestos and arsenic.3 In addition,
hyperten-sion is a known risk factor for RCC,4while moderate
alcohol consumption, coffee drinking and tea drink-ing are associated with a decreased risk of RCC.5
In a study on arsenic, one of the most significant hazards in the environment, Chen et al reported higher age standardized mortality in kidney cancer in the blackfoot disease endemic area.6Tan et al did
not observe an association of high arsenic levels in drinking water with RCC, and concluded that the carcinogenicity of arsenic may be cell type specific for UC but not for RCC.7 Based on these findings,
the role of arsenic exposure in RCC needs to be clarified.
Huang et al conducted a 12-year followup study in the blackfoot disease endemic area and found that participants with high chronic arsenic exposure and with inefficient arsenic methylation had a higher risk of UC.8 Pu et al reported that subjects with an
unfavorable urinary arsenic profile have an in-creased UC risk even at low arsenic exposure lev-els.9 These studies demonstrated that UC risk was
influenced by arsenic methylation profiles in high and low arsenic exposure areas. Our recent study demonstrated that urinary total arsenic levels were significantly associated with chronic kidney disease, which is defined as an eGFR less than 60 ml/minute/ 1.73 m2in a dose-response relationship.10Renal
tu-mors are common in the pre-transplant end stage renal disease population.11However, to our
knowl-edge until now there was no epidemiological study to verify the relationship between individualized arse-nic methylation capacity and risk of RCC. We clar-ified the association between arsenic methylation capacity and RCC risk, and explored whether low eGFR or hypertension modifies arsenic induced RCC risk even in a low arsenic exposure area.
MATERIALS AND METHODS
Study Subjects and Questionnaire Interview A total of 132 patients with pathologically proven RCC were diagnosed and recruited from the Department of Urology, National Taiwan University Hospital, between November 2006 and May 2009. Pathological verification of RCC was completed by image guided biopsy or surgical resection of renal tumors. A total of 260 age and sex matched control subjects with no evidence of RCC or any other malignancy were collected from those undergoing senior citizen health examinations at Taipei Medical Uni-versity Hospital and those undergoing adult health exam-inations at Taipei Municipal Wan Fang Hospital. All 3 hospitals are located in Taipei, approximately 200 to 300 km away from the arsenic contaminated areas in Taiwan. None of the RCC cases or controls came from these areas. The majority of study participants (greater than 80%)
lived in Taipei City and drank tap water from the Taipei Water Department of the Taipei City Government. The average arsenic concentration of tap water in Taipei City is 0.7g/l but concentrations range from nondetectable to 4.0g/l.
Well trained personnel performed standardized per-sonal interviews based on a structured questionnaire. In-formation collected included demographic and socioeco-nomic characteristics, general potential risk factors for malignancies such as lifestyle, consumption of alcohol, tea and coffee, and cigarette smoking in quantified detail, as well as histories of hypertension and diabetes. Frequent alcohol, tea and coffee drinkers referred to those who consumed the respective drinks 2 or more days per week for at least 6 months. Those who consumed less than this level were classified as occasional drinkers. The Research Ethics Committee of National Taiwan University Hospital approved the study. All patients signed informed consent forms before sample and data collection.
Daytime midstream urine samples and serum samples were collected. Serum creatinine was measured after pa-tient diagnosis but before surgery or treatment. Urine was stored at⫺20C until further use for urinary arsenic spe-ciation. Urinary creatinine is almost universally used to adjust concentrations of urinary analytes for variations in hydration status. Therefore, we adjusted urinary total arsenic concentration with urinary creatinine (mg/dl). Ac-cording to a report comparing the concentration of arsenic in individual urine voids with arsenic in a 24-hour urine collection, the concentration of arsenic in urine is stable throughout the day.12Therefore, the use of spot urine
collec-tion to reflect 24-hour excrecollec-tions for arsenic may be reliable. The TNM classification of the American Joint Committee on Cancer was used for pathological staging.13Tumor grading
was based on the Fuhrman grading system.14eGFR is
tra-ditionally considered the best overall index of renal function in health and disease. We used the abbreviated equation from the Modification of Diet in Renal Disease Study15 to
estimate eGFR as 186.3 ⫻ (serum creatinine)⫺1.154 ⫻ (age)⫺0.203⫻ (0.742 for female).
Determination of Urinary Arsenic Species
Frozen urine samples were thawed at room temperature, dispersed by ultrasonic wave and filtered through a Sep-Pak® C18 column. A urine aliquot of 200l was used for the determination of arsenic species by high performance liquid chromatography (Waters 501, Waters Associates, Milford, Massachusetts) with columns obtained from Phe-nomenex (Nucleosil, Torrance, California). The contents of AsIII, AsV, MMAVand DMAVwere quantified by hydride generator atomic absorption spectrometry.16The method
of arsenic speciation is not influenced by dietary factors such as the ingestion of shellfish, fish or other seafood.17
Recovery rates of the 4 arsenic species were calculated by [(sample spiked standard solution concentration) - sample concentration]/standard solution concentration⫻ 100. Re-covery rates for AsIII, AsV, MMAV and DMAV ranged between 93.8% and 102.2%, with detection limits of 0.02, 0.06, 0.07 and 0.10g/l, respectively. Urinary concentra-tion of the sum of inorganic arsenic, MMAVand DMAV, was named total arsenic. The standard reference mate-rial, SRM 2670, contained 480 ⫾ 100 g/l inorganic
arsenic (National Institute of Standards and Technology, Gaithersburg, Maryland), and was used as a quality stan-dard and analyzed along with urine samples. The mean concentration of SRM 2670 determined by our system was 507⫾ 17g/l (n ⫽ 4).
Statistical Analysis
Student’s t test was used to compare differences in the urinary arsenic profiles between case subjects and con-trols. ANOVA and Scheffe’s multiple comparison were applied to compare urinary arsenic profiles between var-ied exposure strata. Multiple logistic regression models were used to estimate the multivariate adjusted odds ra-tios and 95% CIs. Cutoff points for continuous variables were the respective tertiles of distribution in controls. Significance tests for linear trends among ORs across ex-posure strata were calculated by categorizing exex-posure variables and treating scored variables as continuous. For the joint effect analysis the cutoff point for the urinary total arsenic or percentage of arsenic species were the respective medians of the controls. The joint effects of urinary total arsenic and hypertension on RCC risk were evaluated on multiplicative and additive scales. The bi-nary interaction terms were calculated by multiplying the indicators for 2 explored risk factors and were added to the main effect models. Their significance was then tested by the likelihood ratio statistic based on a multiplicative model. The OR values and their variance covariance ma-trix were then used to calculate values for synergy index and 95% CIs.18SAS® version 8.2 was used for all
statis-tical analyses.
RESULTS
The pathology characteristics revealed a distribu-tion of the types of RCC cases as clear cell type 109, papillary type 9, chromophobe 10 and other types 4, similar to the previous study.19Participants with a
lower eGFR had a significantly higher risk of RCC than those with a higher eGFR in a dose-response relationship after multivariate adjustment. Occa-sional alcohol, tea and coffee drinkers had a
signif-icantly lower RCC risk than nondrinkers. In con-trast, subjects who were former cigarette smokers had a significantly higher OR of RCC than those who had never been smokers after multivariate ad-justment. Hypertension was significantly related to RCC after adjustment for other risk factors.
Urinary total arsenic was marginally higher in cases than in controls (25.16⫾ 2.22 vs 21.15 ⫾ 1.02 g/l, p ⫽ 0.09). Urinary total arsenic level was sig-nificantly associated with risk of RCC in a dose-response relationship after multivariate adjust-ment. Arsenic methylation indices did not show any relationship with RCC risk.
The log-transformed urinary total arsenic level was significantly negatively associated with eGFR after adjusting for multiple covariates (see figure). Hypertension or eGFR tended to interact multipli-catively with total urinary arsenic level in modifying RCC risk. The additive interactions were statisti-cally insignificant.
The risk of RCC was estimated for each combina-tion of hypertension, eGFR and urinary total arsenic using nonhypertension, eGFR 60 ml/minute/1.73 m2
or greater and urinary total arsenic 15.95g/g cre-atinine or less as the reference group (seetable). The greatest odds ratio (6.01) was seen in the subjects with hypertension, eGFR less than 60 ml/minute/ 1.73 m2and urinary total arsenic greater than 15.95
g/g creatinine. A trend test indicated that the risk of RCC increased along with the accumulating num-ber of these 3 risk factors (p⬍0.0001).
DISCUSSION
This study demonstrated that urinary total arsenic levels were associated with RCC risk in a dose-response relationship after adjustment for multi-ple risk factors. Participants with hypertension or with a low eGFR had a significantly increased risk of RCC compared to those with nonhypertension or a high eGFR after adjusting for multiple risk factors.
Association between log transformed urinary total arsenic and eGFR.
Adjusted odds ratios of RCC by hypertension, eGFR and urinary total arsenic
Risk Factors* No. Cases/Controls Age-Gender Adjusted OR (95% CI) Multivariate Adjusted OR (95% CI)† None 22/91 1.00 1.00 1 59/106 2.63 (1.46–6.45)‡ 2.60 (1.37–4.94)‡ 2 34/45 3.87 (1.95–7.67)§ 4.36 (2.01–9.46)§ 3 17/17 5.70 (2.36–16.04)§ 6.01 (2.26–16.04)§ * Hypertension status, eGFR less than 60 ml/minute/1.73 m2and urinary total
arsenic greater than 15.95g/g creatinine.
† Adjusted for age, gender, diabetes, body mass index, tea or coffee drinking and cumulative cigarette smoking.
‡ p⬍0.01. § p⬍0.001.
Urinary total arsenic levels of participants in this study were significantly lower than those in our previous study, which included skin cancer cases and healthy controls from the arsenic contaminated area in southwestern Taiwan (104.1 ⫾ 15.2 and 89.5 ⫾ 7.2 g/l, respectively).16 Nevertheless, the
total arsenic levels reported here (RCC cases 25.2⫾ 2.2, controls 21.4⫾ 1.0g/l) suggest that subjects in this study had been exposed to low levels of arsenic. We still observed an increased RCC risk in subjects with high urinary total arsenic, which is the same result seen in our previous studies.9,10
Absorbed arsenic undergoes complicated bio-methylation in the liver to form MMAVand DMAV that are excreted by the kidneys into the urine.20
This pathway is the major pathway for the metabo-lism of inorganic arsenic in humans, followed as AsV
¡ AsIII ¡ MMAV ¡ MMAIII ¡ DMAV, and involves reduction and oxidative methylation. Re-duction is catalyzed by purine nucleoside phosphor-ylase or glutathione s-methyltransferase omega, and oxidative methylation is catalyzed by arsenite/ MMAIII methyltransferase.21 MMAIII and DMAIII have been identified in human urine22 and many
studies have demonstrated that those arsenic spe-cies are more toxic than the inorganic compounds.23 However, the trivalent methylated arsenic metabo-lites are not stable, and whether they can be de-tected depends on the conditions of sample storage and concentrations in the urine. We did not observe any trivalent methylated metabolites in this study because the analytical method used lacks the requi-site specificity. In general, arsenic methylation is considered a detoxification process in which MMAV
and DMAV are considered nontoxic. In the present study we only found that urinary total arsenic but not methylation capability was significantly associ-ated with RCC risk, although subjects ingested low levels of arsenic in drinking water.
The inorganic arsenic percentage and DMA per-centage was significantly different between stages I and II, and stages I and III, and there was no asso-ciation between the other arsenic indices and tumor stage, grade, cell type and tumor size (data not shown). Therefore, these results may indicate that arsenic methylation capability may not affect RCC prognosis.
The major function of the kidneys is to regulate systemic water and electrolyte balance, and excrete waste products and foreign chemicals. Many studies have reported that the relative risk of RCC in pa-tients with end stage renal failure is estimated to be 5 to 20-fold higher than that of the general popula-tion.11 In this study hypertension was related to RCC, and the eGFR of patients with hypertension was significantly lower than in those without
hyper-tension (data not shown). However, a lower eGFR was associated with a higher risk of RCC and eGFR was also significantly negatively related with log-transformed urinary total arsenic. These results may suggest that urinary total arsenic induced hy-pertension24 or influenced the kidney function10to modify the risk of RCC, and this association needs further examination. The mechanism by which hy-pertension contributes to RCC is unclear, but it may be associated with related functional or metabolic changes such as decreased blood flow to the kidney and cell proliferation in the renal tubule that can subsequently induce hypoxia and promote angiogen-esis.25
In this study urinary total arsenic was signifi-cantly related to the risk of RCC, possibly through mechanisms or modes of action for arsenic carcino-genesis, including oxidative stress. Inorganic arse-nic induced oxidative damage results in chroarse-nic pathological states in the kidney that involve reac-tive oxygen species production.26However, oxidative stress has been identified as an important mecha-nism in arsenic induced decreased kidney function through accumulation of arsenic in kidney tissue, increased levels of serum urea nitrogen and lipid peroxidation end products, and reduced glutathione in a mouse model.27Our recent study showed that arsenic methylation species were associated with oxidative damage, which was assessed using uri-nary 8-hydroxydeoxyguanosine,28 suggesting that arsenic metabolites are related to oxidative stress. On the other hand, the strong local electronic mo-lecular interactions between arsenic ions and the membrane bound neural endopeptidase CD1029that
might induce arsenic hydrate microcrystals might also be involved in arsenic induced carcinogenicity in terms of the development of renal malignancy.30
These mechanisms merit further investigation. Our study has some important limitations that need to be considered when interpreting the results. The accuracy of spot evaluation of urinary arsenic species may be in doubt. However, the values might be reliable because none of the subjects experienced a change in lifestyle and appeared to maintain their homeostatic metabolism. In addition, the informa-tion on some risk factors for RCC, including envi-ronmental exposures such as asbestos or advanced kidney disease,3 was unavailable to control in our analyses. Because of the small sample size, statisti-cal significance should be interpreted with caution. Finally, RCC prevalence cases were recruited in this study. However, we cannot exclude the fact that the association between urinary total arsenic levels and RCC in this study might be the result of and not the cause of RCC.
CONCLUSIONS
This is the first study to our knowledge showing that higher urinary total arsenic is significantly associated with an increased risk of RCC. Our data also provided evidence that subjects with
high urinary total arsenic levels may experience an increased risk of RCC if they had hypertension and if they had an eGFR less than 60 ml/minute/ 1.73 m2, even if they ingested low levels of arsenic in drinking water.
REFERENCES
1. Jemal A, Siegel R, Ward E et al: Cancer statis-tics. CA Cancer J Clin 2006; 56: 106. 2. Bureau of Health Promotion: Cancer Registry
An-nual Report, 2006. Department of Health, Execu-tive Yuan, Taiwan, ROC 2006.
3. Pascual D and Borque A: Epidemiology of kidney cancer. Adv Urol 2008; 782381.
4. Chow WH, Gridley G, Fraumeni JF et al: Obesity, hypertension, and the risk of kidney cancer in men. N Engl J Med 2000; 343: 1305. 5. Hu J, Mao Y, DesMeules M et al: Total fluid and
specific beverage intake and risk of renal cell carcinoma in Canada. Cancer Epidemiol 2009; 33: 355.
6. Chen CJ, Kuo TL and Wu MM: Arsenic and cancers. Lancet 1988; 1: 414.
7. Tan LB, Chen KT and Guo HR: Clinical and epi-demiological features of patients with genitouri-nary tract tumour in a blackfoot disease endemic area of Taiwan. BJU Int 2008; 102: 48. 8. Huang YK, Huang YL, Hsueh YM et al: Arsenic
exposure, urinary arsenic speciation, and the in-cidence of urothelial carcinoma: a twelve-year follow-up study. Cancer Causes Control 2008; 19: 829.
9. Pu YS, Yang SM, Huang YK et al: Urinary arsenic profile affects the risk of urothelial carcinoma even at low arsenic exposure. Toxicol Appl Phar-macol 2007; 218: 99.
10. Hsueh YM, Chung CJ, Shiue HS et al: Urinary arsenic species and CKD in a Taiwanese popula-tion: a case-control study. Am J Kidney Dis 2009;
54: 859.
11. Denton MD, Magee CC, Ovuworie C et al: Prev-alence of renal cell carcinoma in patients with ESRD pre-transplantation: a pathologic analysis. Kidney Int 2002; 61: 2201.
12. Calderon RL, Hudgens E, Le XC et al: Excretion of arsenic in urine as a function of exposure to arsenic in drinking water. Environ Health Perspect 1999; 107: 663.
13. American Joint Committee on Cancer: Cancer Staging Manual. Philadelphia: Lippincott-Raven 2002.
14. Fuhrman SA, Lasky LC and Limas C: Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982; 6: 655. 15. National Kidney Foundation: K/DOQI clinical
prac-tice guidelines for chronic kidney disease: evalu-ation, classificevalu-ation, and stratification. Am J Kid-ney Dis 2002; 39: S1.
16. Hsueh YM, Chiou HY, Huang YL et al: Serum beta-carotene level, arsenic methylation capabil-ity, and incidence of skin cancer. Cancer Epide-miol Biomarkers Prev 1997; 6: 589.
17. Hsueh YM, Hsu MK, Chiou HY et al: Urinary arsenic speciation in subjects with or without restriction from seafood dietary intake. Toxicol Lett 2002; 133: 83.
18. Hosmer DW and Lemeshow S: Confidence inter-val estimation of interaction. Epidemiology 1992;
3: 452.
19. Störkel S, Ebie JN, Adlakha K et al: Classification of renal cell carcinoma: Workgroup No. 1. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997; 80: 987.
20. Thompson DJ: A chemical hypothesis for arsenic methylation in mammals. Chem Biol Interact 1993; 88: 89.
21. De CS, Ghosh P, Sarma N et al: Genetic variants associated with arsenic susceptibility: study of purine nucleoside phosphorylase, arsenic (⫹3) methyltransferase, and glutathione s-transferase omega genes. Environ Health Perspect 2008; 116: 501.
22. Mandal BK, Ogra Y and Suzuki KT: Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected ar-eas in West Bengal, India. Chem Res Toxicol 2001; 14: 371.
23. Petrick JS, Ayala-Fierro F, Cullen WR et al: Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes. Toxicol Appl Pharmacol 2000; 163: 203. 24. Huang YK, Tseng CH, Huang YL et al: Arsenic
methylation capability and hypertension risk in subjects living in arseniasis-hyperendemic areas in southwestern Taiwan. Toxicol Appl Pharmacol 2007; 218: 135.
25. Chi JT, Wang Z, Nuyten DS et al: Gene expres-sion programs in response to hypoxia: cell type specificity and prognostic significance in human cancers. PLoS Med 2006; 3: 395.
26. Sasaki A, Oshima Y and Fujimura A: An approach to elucidate potential mechanism of renal toxicity of arsenic trioxide. Exp Hematol 2007; 35: 252. 27. Sinha M, Manna P and Sil PC: Arjunolic acid
attenuates arsenic-induced nephrotoxicity. Patho-physiology 2008; 15: 147.
28. Chung CJ, Huang CJ, Pu YS et al: Urinary 8-hy-droxydeoxyguanosine and urothelial carcinoma risk in low arsenic exposure area. Toxicol Appl Pharmacol 2002; 226: 14.
29. Pan CC, Chen PC and Ho DM: The diagnostic utility of MOC31, BerEP4, RCC marker and CD10 in the classification of renal cell carcinoma and renal oncocytoma: an immunohistochemical anal-ysis of 328 cases. Histopathology 2004; 45: 452. 30. Gunia S: Molecular interaction between arsenic hydrate microcrystals and the cell-surface endo-peptidase CD10 (neprilysin) - a possible link to the development of renal and cutaneous malig-nancies upon occupational exposure to arsenic compounds? Med Hypotheses 2010; 75: 35.
