1 Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.
2 Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA.
3 Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, IA.
4 Department of Civil and Environmental Engineering, University of Iowa, Iowa City, IA.
Received for publication August 28, 2003; accepted for publication April 8, 2004.
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ABSTRACT |
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arsenic; case-control studies; melanoma; skin neoplasms
Abbreviations: Abbreviations: CI, confidence interval, OR, odds ratio
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INTRODUCTION |
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Arsenic is a naturally occurring metalloid element. Commercial use of arsenical compounds in various industries was common (2, 3) but has declined in more recent years (4). Water contamination can occur naturally when arsenic-rich ores leach into ground- and surface water (5). Some areas in Iowa have high levels of arsenic in the water supplies. One survey by the Iowa Department of Natural Resources estimated that up to 12 percent of Iowas municipal water supplies include wells or sources of water with arsenic concentrations greater than or equal to 10 µg/liter (6). In this subset of supplies with high concentrations, the highest concentration detected was 80 µg/liter and the average concentration was 21 µg/liter. The arsenic concentration in private wells is less well characterized because there are no regulatory databases to capture this information. A recent US Geological Survey project sampled wells and compiled data to estimate arsenic concentrations in groundwater, including private wells. Because of insufficient data for Iowa, it is not possible to estimate the arsenic concentration in groundwater by using this database (7).
A number of epidemiologic studies and case reports link arsenic with the development of skin cancer (3, 811). Many of these studies were based on ecologic data from Taiwan, where levels of arsenic were much higher (10, 12) than most estimates in the United States (4). Extrapolation from risk assessments of highly exposed populations has indicated that arsenic levels as low as 2 µg/liter may be carcinogenic (13). A New Hampshire study (9) attempted to quantify exposure to arsenic in relation to development of nonmelanoma skin cancer. The authors reported odds ratios of 2.07 (95 percent confidence interval (CI): 0.92, 4.66) and 1.44 (95 percent CI: 0.74, 2.81) for squamous cell carcinoma and basal cell carcinoma, respectively, for the highest category of arsenic (9). To our knowledge, there have been no studies of arsenic exposure and melanoma incidence, although ecologic data and mortality studies have suggested a potential link between elevated arsenic levels and melanoma (2, 14, 15).
Arsenic exposure may come from a variety of sources; biomarkers that represent a persons recent total arsenic exposure are superior to measurement from a single source. Arsenics affinity for sulfhydryl groups of keratin causes accumulation where scleroproteins are abundant, such as hair, fingernails, and toenails, which can then be used to quantify a persons exposure. Toenail clippings are an excellent biomarker because they are less susceptible than hair to external contamination, are easy to collect and maintain, and represent long-term exposure (312 months prior to collection) (16, 17).
The goal of this case-control study was to examine arsenic content in toenails in relation to cutaneous melanoma.
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MATERIALS AND METHODS |
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Both cases and controls were restricted to those diagnosed with malignant cancer and alive at the time of survey. For comparability, both groups were additionally restricted to Whites aged 40 years or older, since melanoma is predominantly a disease of Whites and few colorectal cancer patients were younger than age 40 years. Of 1,395 melanoma cases diagnosed, 662 met inclusion criteria. Since there were more colorectal cancer cases who met these criteria (n = 2,500), the control group was sampled on the basis of gender and 5-year age group, frequency matching at a one-to-one case-to-control ratio. Not as many persons were diagnosed with colorectal cancer as melanoma between ages 40 and 49 years, so colorectal controls aged 5059 years were oversampled, effectively making the bottom age stratum for frequency matching age 4059 years. Additionally, we oversampled controls by 15 percent because of the lower survival rates associated with colorectal cancer. Of the eligible colorectal cancer controls, we randomly selected 776 who met inclusion criteria for contact.
After cases and controls were identified, a letter was sent to the patients physician asking whether there was a reason that the patient should not be contacted, such as severe illness, mental incompetence, or death. If the physician did not respond within 3 weeks, it was considered passive physician consent to contact the patient. Then, a copy of the survey, a cover letter outlining the study, a toenail collection kit, and an informed consent document were sent to cases and controls. If no response was received within 3 weeks, a reminder letter was sent again asking for participation. After another 36 weeks with no response, an attempt was made to contact the subject by telephone. On average, five attempts were made at different times of the day to contact each person for whom a telephone number could be ascertained. Subjects not contacted by telephone, and those contacted who agreed to participate but had misplaced their surveys, were sent another survey and toenail collection packet. The University of Iowas Institutional Review Board approved this recruitment protocol and all study materials.
Of 662 melanoma cases and 776 colorectal cancer controls initially selected, 12 melanoma patients and 31 colorectal cancer patients were reported deceased by either their physician or a family member. For an additional 15 patients (five melanoma, 10 colorectal), the physician requested that the patient not be contacted because of reasons such as dementia, mental retardation, incarceration, and severe illness. Three controls were removed from our study after completing their surveys because they indicated that their race was other than White (one Asian, two Native American).
Of 645 eligible melanoma cases, 368 responded to the survey (57.1 percent) and 355 provided toenail clippings (55.0 percent). Of 732 eligible colorectal cancer patients, 373 returned the survey (50.9 percent) and 353 submitted toenail clippings (48.2 percent). Overall, of those who returned the survey, 95.5 percent also returned toenail clippings.
Information on eligible nonrespondents was obtained from Iowa Cancer Registry records. Respondents and nonrespondents were similar with respect to gender and stage at diagnosis of their current cancer. They were also similar regarding whether they lived in urban or rural areas (p = 0.3). However, respondents were younger than nonrespondents and were more likely to be married. Respondents from both case and control groups were as likely as nonrespondents to have had a prior cancer diagnosis, and that prior cancer diagnosis was more likely to be malignant (compared with in situ) for both groups.
Arsenic exposure assessment
Study participants collected and submitted toenail clippings in provided, prelabeled, plastic bags. Samples were sent to the Exposure Assessment Facility Core of the University of Iowas Environmental Health Sciences Research Center, where they were washed in acetone to remove dirt and nail polish and were weighed on a microbalance. The weight of samples ranged from 7.2 mg to 855.4 mg (mean, 94.1 mg). Toenail clippings were digested in a nitric acid solution and were placed in a 95°C incubator for 30 minutes or until digestion was complete. Digested samples were analyzed by using graphite furnace atomic absorption spectrophotometry. The instrument used was a Perkin-Elmer 3300 with HG600 graphite furnace, AS90 autosampler, and an EDL2 external lamp power supply (Perkin-Elmer, Wellesley, Massachusetts).
Samples were compared against a reagent blank and an arsenic standard. The arsenic standard was prepared by using a 1,000-mg/liter arsenic standard solution (Perkin-Elmer). This standard was diluted with 10 percent nitric acid to make a 25-µg/liter solution, which was diluted by the instrument to create a five-point calibration curve ranging from 1.25 µg/liter to 25 µg/liter. The minimum detectable level of arsenic using this method was approximately 2.5 µg/liter. For a 94.1-mg sample, this level corresponds to 0.027 µg of arsenic per gram of toenail.
Statistical analyses
Descriptive analyses were performed for demographic study variables for cases and controls, including frequency distributions and other summary statistics. Log-transformed toenail arsenic concentrations were normally distributed. Cutpoints were set based on quartiles of arsenic concentration in controls. Arsenic was also considered as an ordered categorical variable to test for linear trend. It was presumed that subjects were exposed primarily through their residential water; 44 participants who had changed residences since their diagnosis were excluded from these analyses to reduce misclassification of exposure.
The arsenic content of some toenail samples was below the analytical limit of detection (n = 304), posing the common problem of a left-censored log-normal distribution. Since the actual arsenic concentrations have values between zero and the detection limit, we imputed values for these samples by assigning them the minimum detectable limit divided by the square root of 2, a method often referred to as triangular approximation (18).
Unconditional logistic regression was used to examine melanoma in relation to toenail arsenic levels (19). All analyses controlled for any residual confounding due to age, gender, and education. For arsenic content, we assessed effect modification by history of sunburn, prior cancer diagnosis, prior skin cancer diagnosis, and time at the current residence. If no effect modification was seen, potential confounding was assessed for these factors as well as for skin color and skin type. Confounding was determined by a 10 percent or more change in the odds ratio.
Residential water source and occupation were explored as potential sources of exposure. For those who used private wells as their primary source of water, well depth was considered with respect to arsenic concentration. Occupations in which subjects worked with wood treated with chromium copper arsenate or those potentially involved in arsenical pesticide production and application industries were considered at risk for occupational exposure to arsenic (4). Industries that traditionally involve high arsenic exposures, such as copper smelting, are not common in Iowa (4). Because of the low prevalence of these occupations and because we did not measure actual arsenic levels on the job, we classified participants as potentially exposed to arsenic if they reported employment in industries that had the possibility for arsenic exposure, such as farming, carpentry, construction, golf course maintenance, or lumber yard work. Participants with a high arsenic toenail concentration were compared with those with a low concentration based on their employment in these fields and with respect to self-reported occupational arsenic exposure.
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RESULTS |
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The association between toenail arsenic concentration and melanoma showed a significant increasing linear trend with increasing toenail arsenic concentration (table 2). The odds ratio for the highest quartile of arsenic concentration compared with the lowest was 2.1 (95 percent CI: 1.4, 3.3). No confounding by skin color, skin type, or prior history of sunburn was found. We did see a significant effect modification between arsenic content and risk of melanoma by self-reported prior skin cancer diagnosis (table 3). Risk of melanoma with increasing toenail arsenic content was much greater for those with a prior skin cancer diagnosis (OR = 6.6, 95 percent CI: 2.0, 21.9) than for those without (OR = 1.7, 95 percent CI: 1.0, 2.8). When we stratified by time at the current residence (table 4), elevated odds ratios were found for the highest arsenic exposure category for those who had lived at their current residence less than 15 years (OR = 2.8, 95 percent CI: 1.4, 5.8) as well as for those who had lived there for 15 years or more (OR = 1.8, 95 percent CI: 1.0, 3.4). Similar results were found when stratifying by less than 10, 1019, and 20 years or longer at the current residence.
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DISCUSSION |
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The use of a biomarker accounting for arsenic from all sources eliminates the need for participants to recall their exposures, thereby reducing the potential for recall bias. However, collection 23 years after diagnosis presents a limitation of this technique in a case-control study. We cannot discount the possibility that misclassification of exposure to arsenic occurred because of changing exposures postdiagnosis. In this study, the average length of time that subjects lived at their current location was 21.9 years. If the latency period for arsenic is shorter than 20 years, and if people are exposed primarily through their drinking water, it seems likely we would have captured the relevant period of arsenic exposure. We stratified our analyses to investigate a potential difference in risk based on time at the current residence. We observed no effect modification by time at the current residence and found elevated odds ratios for the highest exposure in both time periods. The mean length of time at the current residence was over 20 years. However, it is possible that the source of water could have changed while residence remained constant, meaning that arsenic exposure through drinking water could be more variable than suggested by time at the current residence.
Increased postdiagnosis arsenic levels in melanoma cases are unlikely to be related to treatment. The preferred course of treatment for melanoma is surgical excision (21, 22). Chemotherapeutic drugs for melanoma treatment include dacarbazine, interferon, cisplatin, tamoxifen, and carmustine (23, 24). These therapies are not known to contain arsenic, making it unlikely that increased arsenic levels in melanoma cases are due to treatment.
When compared with subjects using community supplies as their water source, those using private wells were more likely to have toenail arsenic concentrations in the highest quartile than in the lowest. Private wells are not subject to the same requirements for testing as are public water supplies, leading to the possibility of undetected contamination. In a New Hampshire study, private wells were associated with higher levels of arsenic than were public water supplies (25). That study also showed a correlation between arsenic levels in the residential water supply and toenail arsenic concentrations. The amount of arsenic in the toenail that corresponds to particular concentrations in drinking water is unknown and likely depends on the amount of water consumed and exposure to other sources of arsenic. In the New Hampshire study, 1 µg of arsenic per liter of water corresponded to approximately 0.1 µg of arsenic per gram of toenail. A doubling of toenail arsenic concentration was associated with a 10-fold increase in water arsenic in those samples at or above 1 µg/liter (25).
Although water supplies are presumed to be the most common means by which study participants are exposed to arsenic, occupational exposures may be important in a subsample of this population. In our study, over twice as many subjects in the highest quartile of arsenic exposure had reported a known occupational exposure to arsenic than those in the lowest quartile. Additionally, those classified as being in higher risk jobs were more likely to be in the highest quartile of arsenic concentration than in the lowest.
This study had several strengths. Cases and controls were ascertained through the Iowa Cancer Registry, a Surveillance, Epidemiology, and End Results Program registry. This registry enabled population-based ascertainment of newly diagnosed melanoma cases in a specified time period and a high degree of certainty about accuracy of diagnosis. Colorectal cancer controls were selected from the same registry and came from the same underlying population. Toenail arsenic levels are not susceptible to recall bias and are used to estimate total body burden. Concentration has been shown to be relatively stable; a reproducibility study of trace elements in toenails found arsenic levels to be highly correlated over a 6-year follow-up (26).
Our study found an increased risk of melanoma with history of sunburn and sun sensitivity factors, with observed odds ratios in the range of 1.42.9, which concurs with other studies of conventional risk factors for melanoma (2732). This similarity of findings lends credibility to other results we found concerning melanoma and arsenic.
The primary limitation of this study is comparison of melanoma cases with cancer controls. Cancer controls were chosen because of the difficulty in ascertaining appropriate population-based controls. Use of drivers license records has traditionally been a good way to identify controls (33), but this method has changed recently because of a federal law restricting access to department of motor vehicle records for research purposes (34). Limited funding also played a role in our selection of colorectal cancer controls. We are unaware of any studies linking colorectal cancer incidence to arsenic exposure. We were unable to find literature suggesting that arsenic absorption was affected either by colorectal cancer itself or by common treatment drugs. We were also unable to find any evidence that the disease or its treatment affected toenail loss. In our analyses, we saw no differences in toenail arsenic concentration by treatment. One early study did report an association of arsenic exposure with colorectal cancer mortality (8), but several other studies have failed to find a similar increase, although these studies were smaller in number than the original (3539). We chose to use only one cancer site as a control group because of the difficulty identifying another cancer site that was not associated with arsenic or sun exposure, included an adequate number of cases, and had a relatively good survival rate since we were contacting people 23 years after diagnosis.
Another concern with this group was the older age of the controls. Since age was not correlated with arsenic concentration, this age difference was probably not a factor in the observed association with arsenic. Although we frequency matched on gender when recruiting participants for this study, males in the control group were more likely to participate than were females, resulting in a greater number of females in the case group than in the control group. Arsenic concentration was not correlated with gender.
An additional drawback of using cancer controls is the possibility that risk factors of interest are also associated with disease in controls. If this scenario were to occur, it would bias results toward the null. For our results to be biased because of use of colorectal cancer cases, colorectal cancer would have to be inversely associated with arsenic exposure. Arsenic is recognized as a human carcinogen; therefore, it is unlikely to be protective for colorectal cancer (40).
The relatively low response rate (53.2 percent overall) is another limitation of this study. This limitation could have led to nonresponse bias if respondents had different exposures than nonrespondents, which could bias results in either direction. For arsenic, there is no reason to believe that respondents and nonrespondents were more or less likely to be exposed. According to the Iowa Department of Natural Resources, the percentage of Iowans using some source other than public supplies for their water was comparable between our study (15.8 percent) and the Iowa general population (14.2 percent), indicating that our control group was similar to the general population (41). Additionally, respondents and nonrespondents were similar with respect to living in urban or rural areas. Therefore, nonresponse bias should be less of a concern for these analyses. Nonrespondents were significantly older than respondents in this study. There was potential for survival bias, since participation in the study was restricted to those people still living. This bias could occur if the exposure of interest is related to a more virulent disease process, thereby causing death from melanoma at a more rapid rate than would otherwise occur. Exclusion of these cases would therefore have biased results toward the null, and the association found here would likely be an underestimate of the true magnitude. Conversely, in the unlikely event that arsenic is associated with a more virulent form of colorectal cancer, gathering information from only living cases would result in an overestimation of the association with melanoma.
In summary, to our knowledge, the association between increasing arsenic exposure and cutaneous melanoma risk has not been previously reported and is important because of the potential for large numbers of people to be exposed to arsenic. We observed an even higher effect among those with a prior nonmelanoma skin cancer diagnosis, which lends further support to a causal association between arsenic and cutaneous melanoma. In Iowa, use of private wells for residential drinking water appeared to be associated with both increased toenail arsenic content and increased melanoma risk. While it appears that water supplies are the means by which most persons are exposed to arsenic, the possibility of occupational exposure to arsenic cannot be excluded. The association we observed with arsenic is not known to have been previously reported in observational studies of incident cutaneous melanoma. Therefore, the findings warrant confirmation.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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