ARTICLE

Alkylaniline–Hemoglobin Adducts and Risk of Non–Smoking-Related Bladder Cancer

Jinping Gan, Paul L. Skipper, Manuela Gago-Dominguez, Kazuko Arakawa, Ronald K. Ross, Mimi C. Yu, Steven R. Tannenbaum

Affiliations of authors: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA (JG, PLS, SRT); Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles (MGD, KA, RKR, MCY)

Correspondence to: Paul L. Skipper, PhD, Massachusetts Institute of Technology, Bldg. 56, Rm. 753, 77 Massachusetts Ave., Cambridge, MA 02139 (e-mail: skipper{at}mit.edu)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Some members of the arylamine family of compounds, specifically 4-aminobiphenyl (ABP), 2-naphthylamine, and benzidine, are established human bladder carcinogens. Cigarette smoking and use of permanent hair dye contribute substantially to current arylamine exposure. Low levels of 4-ABP exposure have been associated with non–smoking-related bladder cancer. Other arylamine compounds coming from as yet unidentified environmental sources may also be human bladder carcinogens. Methods: We conducted a population-based case–control study in Los Angeles County, California, involving 298 case subjects with bladder cancer and 308 control subjects, who were matched on age, sex, race/ethnicity, and neighborhood of residence. In-person interviews provided information on tobacco smoking and other potential risk factors for bladder cancer. To assess arylamine exposure, levels of arylamine–hemoglobin adducts of nine selected alkylanilines (2,3-dimethylaniline [2,3-DMA], 2,4-DMA, 2,5-DMA, 2,6-DMA, 3,4-DMA, 3,5-DMA, 2-ethylaniline [2-EA], 3-EA, 4-EA) were measured in peripheral blood collected from study subjects. Analysis of covariance and conditional logistic regression methods were used to analyze the relationship between arylamine–hemoglobin adducts and bladder cancer risk. All statistical tests were two-sided. Results: Levels of all arylamine–hemoglobin adducts, with the exception of 2,6-DMA, were higher in smokers than in nonsmokers, and levels of all arylamine–hemoglobin adducts were higher in case subjects than in control subjects. Arylamine–hemoglobin adducts of 2,6-DMA, 3,5-DMA, and 3-EA were all independently, statistically significantly (all P<.001) associated with bladder cancer risk after adjusting for cigarette smoking at the time of blood collection, lifetime smoking history, and other potential risk factors. These adducts were also independently associated with bladder cancer risk when only nonsmokers at time of blood draw were considered (highest quartile versus lowest quartile: 2,6-DMA, relative risk [RR] of bladder cancer = 8.1, 95% confidence interval [CI] = 3.6 to 18.0; 3,5-DMA, RR = 2.7, 95% CI = 1.2 to 6.0; 3-EA, RR = 4.3, 95% CI = 1.6 to 11.6). Conclusions: Diverse arylamine exposures are strongly associated with bladder cancer risk among nonsmokers. Because arylamines may account for a substantial proportion of bladder cancers among the general population, identification of environmental sources of these compounds is needed.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Several members of the arylamine family, which is extensive, have been classified and regulated as human bladder carcinogens. These include 4-aminobiphenyl (4-ABP), 2-naphthylamine, and benzidine. However, other arylamines such as o-toluidine (1) and 2,6-dimethylaniline (2) may also be potential human bladder carcinogens.

Exposure to arylamines, which can occur from many different sources, can be detected with sensitive and specific biomarker measurements (3). Arylamines are oxidized in vivo to N-hydroxylamines, which react with hemoglobin to form adducts that may persist for as long as the adducted hemoglobin remains in circulation. Consequently, quantitative analysis of adducts in a blood sample can be used to assess exposures that occurred during the several months preceding blood collection (4). Because oxidation of arylamines to N-hydroxylamines is generally regarded as a critical bioactivation step (5,6), measuring arylamine–hemoglobin adducts in subjects may be more informative than questionnaire-based exposure assessments, which are often used in epidemiologic studies.

Cigarette smoking, an established risk factor for bladder cancer, is believed to contribute to at least 50% of current bladder cancers cases in the United States (7). Arylamines, including 4-ABP and 2-naphthylamine, are present in tobacco smoke and are the leading putative constituents responsible for bladder cancer development in smokers. Use of hair dye is another substantial source of arylamine exposure in humans, especially among women. We recently reported that there is a dose- and duration-dependent increase in bladder cancer risk among women who use permanent hair dyes and that the increased risk is mainly confined to women who are deficient in arylamine detoxification enzymes (810).

In the Los Angeles Bladder Cancer Study, lifelong nonsmoking case subjects had statistically significantly higher levels of 4-ABP–hemoglobin adducts than lifelong nonsmoking control subjects, suggesting that 4-ABP may be involved in the development of non–smoking-related bladder cancer (11). In this study, which is an extension of the Los Angeles Bladder Cancer Study, we examined the possible relationship between bladder cancer and hemoglobin adducts of nine other commonly occurring and structurally related arylamines.


    SUBJECTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Subjects and In-Person Interviews

Recruitment of subjects in the Los Angeles Bladder Cancer Study has been described in detail (7). Briefly, incident case subjects with bladder cancer were identified through the Los Angeles County Cancer Surveillance Program, which is the population-based Surveillance, Epidemiology, and End Results (SEER)1 program cancer registry of Los Angeles County. Eligible subjects were individuals of non-Asian race/ethnicity between ages 25 and 64 years diagnosed with histologically confirmed cancer between January 1, 1987, and April 30, 1996. Enrolled case subjects (n = 1679) represented approximately 75% of the total number of eligible patients. For each enrolled case subject, an invariant procedure [described in detail in (7)] was followed to recruit a control subject from the same neighborhood of residence as the index case at the time of cancer diagnosis who matched the index case by age (within 5 years), sex, and race/ethnicity (non-Hispanic white, Hispanic white, African-American). A matched control subject could not be found for 5% (n = 86) of enrolled case subjects. For 21 index case patients, the control subject was not matched by race/ethnicity. All study subjects provided written informed consent (separate forms for interview and for donation of blood and/or urine) that had been approved by the University of Southern California Human Subjects Committee. This study was approved by the institutional review boards of the University of Southern California and the Massachusetts Institute of Technology.

In-person, structured interviews were conducted by study personnel in each subject’s home. During the interview, subjects were asked to provide information up to 2 years before the diagnosis of bladder cancer for case subjects or 2 years before the diagnosis of cancer of the index case subject for the matched control subject (i.e., reference year). Information was requested on demographic characteristics, lifetime use of tobacco products and alcohol, usual adult dietary habits, lifetime occupational history, previous medical conditions, and previous medication use.

Beginning in January 1992, all case subjects (n = 1044) and their matched control subjects (n = 979) were asked to provide a blood sample at the end of the in-person interview. We obtained and successfully analyzed blood samples from 761 (73%) case subjects and from 770 (79%) control subjects. All subjects who donated blood were asked detailed questions on their use of tobacco products during the preceding 2 months. All subjects also were asked to collect an overnight urine sample (ending with the first morning void) after consuming two cups of instant coffee (approximately 70 mg of caffeine) in the previous afternoon (between 3 and 6 PM). For subjects who were not coffee drinkers, caffeine tablets were offered as an alternative.

From the 1536 subjects who donated blood, we selected 606 for our study of alkylaniline–hemoglobin adducts. This subset of study participants provided the last 606 samples to be analyzed for 4-ABP–hemoglobin adducts, as described previously (11). (An analytical method to quantify alkylaniline–hemoglobin adducts was not available until after 4-ABP–hemoglobin adduct analysis had been performed on blood samples from the first 930 subjects.) Of the 606 subjects, 298 were case subjects and 308 were control subjects. The study included 190 matched case–control pairs, 108 unmatched case subjects, and 118 unmatched control subjects.

Laboratory Analyses

Blood specimens (10 mL) were collected in heparinized tubes and were fractionated into plasma, buffy coat, and erythrocytes, all of which were stored at –80 °C before analysis. Erythrocytes, identifiable only by their code numbers, were sent on dry ice to the Massachusetts Institute of Technology for arylamine–hemoglobin adduct analysis. Genomic DNA was isolated from buffy coat lymphocytes, and genotypes of glutathione S-transferase M1 (GSTM1), GSTT1, GSTP1, and N-acetyltransferase 1 (NAT1) were determined, as described previously (12,13). NAT2 and CYP1A2 phenotypes were determined by analysis of urinary caffeine metabolites, as described previously (14).

Hemoglobin adducts of nine alkylanilines (2,3-dimethylaniline [2,3-DMA], 2,4-DMA, 2,5-DMA, 2,6-DMA, 3,4-DMA, 3,5-DMA, 2-ethylaniline [2-EA], 3-EA, 4-EA) were subjected to quantitative analysis, as described for the analysis of 4-ABP adducts (15), with the exception that a different internal standard was used and different ions were monitored in the mass spectrometric analysis. The internal standard, [methyl-2H6]-2,6-dimethylaniline-adducted hemoglobin, was prepared according to procedures used for the preparation of the [ring-2H9]-4-ABP hemoglobin adduct internal standard (3). Briefly, [methyl-2H6]-2,6-dimethylnitrobenzene (Isotec, Miamisburg, OH) was reduced with Zn/NH4Cl, and the resulting hydroxylamine was reacted with freshly isolated human hemoglobin. The mixture was then dialyzed against water to remove unbound material, the resulting adducted hemoglobin was diluted to an adduct concentration of approximately 1 ng/mL, and aliquots were stored frozen at –20 °C. The actual concentration of the resulting internal standard was determined by analysis of aliquots spiked with known amounts of isotopically normal 2,6-DMA (Aldrich, Milwaukee, WI).

Samples from the index case subjects and their matched control subjects were always tested in a single batch. For samples from unmatched case subjects or from unmatched control subjects, the number of samples from each group was always similar in a given batch.

Statistical Analysis

The distributions of the alkylaniline–hemoglobin adduct levels in our study population were markedly skewed; therefore, formal statistical testing was performed on logarithmically transformed values of adduct levels, and geometric (as opposed to arithmetic) mean values are presented. Pair-wise Pearson correlation coefficients (on logarithmically transformed values) were calculated for the nine adducts under study to examine the extent to which they were associated. The analysis of covariance method was used to compare adduct levels between case and control subjects, with an adjustment for the effect of cigarette smoking at the time of blood draw on adduct levels (16). The analysis of covariance method also was used to compare adduct levels between exclusive users of permanent hair dyes and nonusers of permanent hair dyes, with an adjustment for the effect of cigarette smoking at the time of blood draw on adduct levels (16). Additional adjustments were also made for other risk factors for bladder cancer identified in the Los Angeles Bladder Cancer Study. These covariates include educational level (17), lifetime history of cigarette smoking (7), regular use of nonsteroidal anti-inflammatory drugs (17), use of permanent hair dyes (810), dietary intake of carotenoids (18), 4-ABP–hemoglobin adducts (11), NAT2 phenotype (unpublished data from this study) and GSTM1 genotype (unpublished data from this study). Results of analyses with or without adjustment for race/ethnicity were similar (data not shown); therefore, race/ethnicity was not retained as a covariate in analyses presented in this study. From all alkylanilines that had a statistically significant univariate association with bladder cancer risk, we used stepwise regression methods to identify the set of alkylanilines that were independent predictors of risk. The backward and forward selection methods yielded the same set of independent risk predictors. Conditional logistic regression models were used to examine the relationship between quartiles of adduct levels and bladder cancer risk. Odds ratios were used to estimate relative risks. Statistical computing was conducted using SAS, version 8.2 (SAS Institute, Cary, NC) and Epilog for Windows, version 1.0 (Epicenter Software, Pasadena, CA) statistical software packages. All P values are two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We determined the correlation coefficients of hemoglobin adduct levels measured for the nine alkylanilines under study (Table 1). With the exception of 2,6-DMA, all alkylaniline–hemoglobin adducts showed positive and relatively close correlations with each other. By contrast, 2,6-DMA adducts showed no association with any of the other alkylaniline adducts. Table 1 also includes the correlation coefficients for 4-ABP–hemoglobin adducts that were previously measured in the same subjects. Correlations between 4-ABP–hemoglobin adducts and any of the alkylaniline–hemoglobin adducts were generally lower than correlations within the set of alkylaniline–hemoglobin adducts, as observed previously (19) in a different population. Analysis of the data reported in (19) suggested that monocyclic arylamines are metabolized by a different set of enzymes than those that metabolize bicyclic arylamines.


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Table 1. Pearson correlation coefficients of alkylaniline–hemoglobin adduct levels*

 
Table 2 presents the geometric means of alkylaniline–hemoglobin adducts among case and control subjects by categories according to subjects’ smoking status. Mean levels of alkylaniline–hemoglobin adducts were higher in smokers than in nonsmokers, with the exception of levels of 2,6-DMA alkylaniline–hemoglobin adducts. In addition, adducts of every alkylaniline were higher in case subjects than in control subjects. Levels of 2,3-DMA–, 2,4-DMA–, 2,6-DMA–, 3,5-DMA–, 3-EA–, and 4-EA–hemoglobin adducts were statistically significantly higher in nonsmoking (at the time of blood draw) case subjects than in nonsmoking control subjects. Among lifelong nonsmokers, levels of 2,6-DMA–, 3,5-DMA–, 2-EA–, 3-EA–, and 4-EA–hemoglobin adducts were statistically significantly higher in case subjects than in control subjects.


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Table 2. Geometric mean (95% confidence interval) levels of selected alkylaniline–hemoglobin adducts, in picograms per gram of hemoglobin, among case subjects with bladder cancer and control subjects*

 
To identify which alkylanilines were independent predictors of the risk of bladder cancer, we next conducted a stepwise regression analysis, with the following covariates always present in the model: age, sex, average number of cigarettes smoked per day during the 2 months before blood draw, educational level, and level of 4-ABP–hemoglobin adducts. From this analysis, three alkylanilines were identified as independent predictors of risk of bladder cancer: 2,6-DMA, 3,5-DMA, and 3-EA (all P<.001).

The relative risks of bladder cancer according to the three alkylanilines that independently predicted the risk of bladder cancer are shown in Table 3. There was a statistically significant increase in risk associated with an increase in adduct levels for each of the three selected alkylanilines. The increased risk persisted when the analysis was restricted to nonsmokers at blood draw. Among nonsmokers at blood draw, individuals in the highest quartiles of 2,6-DMA–, 3,5-DMA–, and 3-EA–hemoglobin adduct levels were three to five times more likely to develop bladder cancer than individuals in the lowest quartiles (relative risk [RR] for 2,6-DMA = 5.5, 95% CI = 2.8 to 10.7; RR for 3,5-DMA = 3.1, 95% CI = 1.6 to 6.0; and RR for 3-EA = 4.8, 95% CI = 2.2 to 10.6). These increased risks persisted after adjustment for other risk factors identified in this study population.


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Table 3. Cut points and relative risks (95% confidence intervals) for bladder cancer associated with alkylaniline–hemoglobin adducts*

 
Previously, we reported that use of permanent hair dye among women is a risk factor for bladder cancer and that use of permanent hair dye among men was relatively uncommon (8). Given that the hair dye–bladder cancer association in women was confined primarily to subjects deficient in arylamine detoxification enzymes, we speculated that the carcinogenic agents in hair dyes might be arylamines (9,10). We therefore examined the levels of nine alkylaniline–hemoglobin adducts according to sex, with adjustment for age at blood draw and smoking status (number of cigarettes smoked per day) at the time of blood draw. Only levels of 3,5-DMA demonstrated a statistically significant difference (P = .04), with levels higher in women than in men (adjusted geometric means, 27.8 pg/g of hemoglobin in women versus 23.2 pg/g of hemoglobin in men). Therefore, we examined 3,5-DMA–hemoglobin adduct levels according to use of permanent hair dye, adjusting for the average number of cigarettes smoked per day during the 2 months before the blood draw. The adjusted geometric mean level of 3,5-DMA–hemoglobin adducts was higher in exclusive users of permanent hair dyes (n = 58) than in nonusers (35.0 pg/g of hemoglobin versus 29.0 pg/g of hemoglobin; P = .08). The difference was statistically significant when the analysis was restricted to women (n = 48 users of permanent hair dyes; 37.9 pg/g of hemoglobin versus 30.4 pg/g of hemoglobin; P = .04). There was no association between use of permanent hair dye and any of the other eight alkylaniline adducts.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
In this population-based investigation of arylamine exposure and bladder cancer risk, case subjects had higher levels of ethyl- and dimethylaniline adducts than their matched control subjects. Most of the differences in adduct levels between case and control subjects were statistically significant, especially among nonsmokers. Adducts of three arylamines—2,6-DMA, 3,5-DMA, and 3-EA—were found to be independent predictors of bladder cancer risk.

Results from toxicologic studies (2) are consistent with a role for 2,6-DMA in human carcinogenesis. 2,6-DMA is a known nasal carcinogen in rats (2) and has been classified by the International Agency for Research on Cancer as a possible human carcinogen (2). Information on the mutagenicity and/or genotoxicity of 3,5-DMA is limited. Although Zimmer et al. (20) and Nohmi et al. (21) reported that 3,5-DMA had no mutagenic activity in various strains of Salmonella typhimurium with or without metabolic activation, Zeiger et al. (22), using the same test, found weak mutagenic activity in the presence of metabolic activation. Little, if any, information is available regarding the potential carcinogenicity of 3-EA.

All of the alkylanilines investigated in this study are known to be present in cigarette smoke (2325). Cigarette smoke is not the only source of alkylanilines, but currently, there is limited information regarding sources other than tobacco smoke for nonoccupational human exposure to arylamines (26). In general, possible additional sources include drugs (27), food (28,29), and azo dyes (30,31). We have been unable to find specific information regarding environmental sources of 3,5-DMA or 3-EA, but there are numerous reports regarding non–smoking-related sources of 2,6-DMA (2,26,27,3235).

2,6-DMA is a metabolite of some drugs (e.g., lidocaine and xylazine) and pesticides (e.g., metalaxyl), and it is an impurity in technical grade metalaxyl (27,3235). Human exposure to 2,6-DMA in patients receiving lidocaine for local anesthesia or cardiac arrhythmias has been inferred from the resulting increase in 2,6-DMA–hemoglobin adduct levels (27). However, the presence of 2,6-DMA–hemoglobin adducts in humans not exposed to lidocaine suggests that exposure to 2,6-DMA comes from other sources (26). 2,6-DMA is a principal metabolite of the veterinary tranquilizer xylazine (36). In the United States, xylazine is not approved by the U.S. Food and Drug Administration (FDA) for use in food-producing animals, but it is unclear whether there is widespread practice of non-FDA approved use or whether imported meats are tested for xylazine (37,38). Canada (the largest import supplier of beef to the U.S. market) and European countries allow use of xylazine in food-producing animals (38). Xylazine residues have been found in bovine milk (36,39) and in different tissues (liver, kidney, site of injection) collected from treated cattle (39).

The Los Angeles Bladder Cancer Study found that use of permanent hair dye by women was a risk factor for bladder cancer, especially among women deficient in genetically controlled arylamine detoxification capacity (810). Consistent with this finding, we found statistically significantly increased levels of 3,5-DMA adducts in women who regularly used permanent hair dyes. There have been no reports, to our knowledge, that 3,5-DMA is an ingredient or a contaminant of commercial brands of permanent hair dyes, but in view of the recent finding (40) that 4-ABP is a contaminant of some hair dyes, it would not be surprising if 3,5-DMA is also.

Previously, we reported that prevalent exposure to the proven human bladder carcinogen 4-ABP is an important risk factor for non–smoking-related bladder cancer (11). In the present study, we have identified 2,6-DMA, 3,5-DMA, and 3-EA as possible additional independent risk factors. Results of the Los Angeles Bladder Cancer Study thus implicate exposure to arylamines as the causal factor responsible for most cases of bladder cancer in humans. Tobacco smoke as a source of these carcinogenic arylamines is already well known. Therefore, identifying the non–smoking-related sources of these carcinogenic arylamines should become a high scientific priority.


    NOTES
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
1SEER is a set of geographically defined, population-based, central cancer registries in the United States, operated by local nonprofit organizations under contract to the National Cancer Institute (NCI). Registry data are submitted electronically without personal identifiers to the NCI on a biannual basis, and the NCI makes the data available to the public for scientific research.

Supported by U.S. Public Health Service grants P01 CA17054, R35 CA53890, and R01 CA65726 from the National Cancer Institute and grants P01 ES05622, P30 ES07048, and P30 ES02109 from the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.

We thank Susan Roberts and Donna Murdock of the University of Southern California for their assistance in the data collection phase of the Los Angeles Bladder Cancer Study and Joe Glogowski of the Massachusetts Institute of Technology for his assistance in processing specimens for hemoglobin adduct analysis.


    REFERENCES
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 Subjects and Methods
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 Discussion
 References
 

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Manuscript received January 22, 2004; revised July 26, 2004; accepted August 5, 2004.


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