1 USC/Norris Comprehensive Cancer Center, Department of Preventive Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033-0800,
2 Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709,
3 Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292 and
4 The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA
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Abstract |
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We also have examined N-acetyltransferase-2 (NAT2) phenotype in relation to the hair dyebladder cancer relationship, and found that the association is principally confined to NAT2 slow acetylators. In the present study, we assessed the possible modifying effects of a series of potential arylamine-metabolizing genotypes/phenotypes (GSTM1, GSTT1, GSTP1, NAT1, NAT2, CYP1A2) on the permanent hair dyebladder cancer association, among female participants (159 cases, 164 controls) of the Los Angeles Bladder Cancer Study. Among NAT2 slow acetylators, exclusive permanent hair dye use was associated with a 2.9-fold increased risk of bladder cancer (95% CI = 1.27.5). The corresponding relative risk in NAT2 rapid acetylators was 1.3 (95% CI = 0.62.8). Frequency- and duration-related doseresponse relationships confined to NAT2 slow acetylators were all positive and statistically significant. No such associations were noted among NAT2 rapid acetylators. Among CYP1A2 slow individuals, exclusive permanent hair dye use was associated with a 2.5-fold increased risk of bladder cancer (95% CI = 1.046.1). The corresponding risk in CYP1A2 rapid individuals was 1.3 (95% CI = 0.62.7). Frequency- and duration-related doseresponse relationships confined to CYP1A2 slow individuals were all positive and statistically significant. No such associations were noted among CYP1A2 rapid individuals. Among lifelong non-smoking women, individuals exhibiting the non-NAT1*10 genotype showed a statistically significant increase in bladder cancer risk associated with exclusive permanent hair dye use (OR = 6.8, 95% CI = 1.727.4). The comparable OR in individuals with the NAT1*10 genotype was 1.0 (95%CI = 0.24.3). Similarly, all frequency- and duration-related doseresponse relationships confined to individuals possessing the non-NAT1*10 genotype were positive and statistically significant. On the other hand, individuals of NAT1*10 genotype exhibited no such associations.
Abbreviations: 4-ABP, 4-aminobiphenyl; CI, confidence interval; DAPPD, N,N'-diacetyl-PPD; GSTs, Glutathione S-transferases; MAPPD, monoracetyl-PPD; NAT2, N-acetyltransferase-2; PPD, paraphenylenediamine
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Introduction |
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This report describes our findings on the potential modifying effect of the different genotypes/phenotypes of enzymes involved in arylamine activation and/or detoxification, including NAT1, NAT2 (extending our earlier results to an expanded data set) (2), GSTM1, GSTT1, GSTP1, and CYP1A2, on the hair dyebladder cancer relationship.
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Subjects and methods |
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For each interviewed case patient, we sought to recruit a control subject who was matched to the index case patient by sex, date of birth (within 5 years), race (non-Hispanic white, Hispanic, AfricanAmerican), and neighborhood of residence at the time of cancer diagnosis. To search for these neighborhood control subjects, we followed an invariant procedure that defines a sequence of houses on specified neighborhood blocks. We attempted to identify the sex, age, and race of all inhabitants of each housing unit; not at home units were systematically revisited to complete the census. The first resident along this defined route who satisfied all eligibility criteria for controls was asked to participate in the study (i.e. first eligible control). If the individual refused, the next eligible control (i.e. second eligible control) in the sequence was asked and so on until we located an eligible control who agreed to be interviewed. When we failed to find any resident who met our matching criteria after canvassing 150 housing units, we excluded race from the matching criteria. If a matched control subject based on the relaxed criteria could not be found within a maximum of 300 housing units, the case patient was dropped from the study. There were six cases for whom a matched control subject could not be found. Thus, a total of 228 female cases and 222 female controls were included in the analysis. All study subjects signed an informed consent form for interview and a separate form for consenting to blood and/or urine donation. The study protocol had been approved by the University of Southern California Institutional Review Board, responsible for protection of human subjects.
In terms of personal hair dye use, we first asked if subject had ever used any type of hair dyes regularly for one year or longer. If the answer was yes, then age started using hair dyes regularly, age stopped using hair dyes regularly, total years of regular use, and frequency of use were recorded. In addition, the types of hair dyes usually used by the subject were noted (permanent, semi-permanent, temporary rinse, permanent and semi-permanent equally, permanent and temporary rinses equally, semi-permanent and temporary rinses equally, all three classes of dyes equally).
Blood sample collection and GSTM1/T1/P1 and NAT1 genotyping
All case patients and their matched control subjects were asked for a blood sample donation at the end of the in-person interview. We obtained a blood sample from 160 of 228 (70%) case patients, and from 164 of 222 (74%) control subjects. Two 10 ml tubes of heparinized whole blood were collected from each study subject. Plasma, buffy coat cells, and red blood cells were isolated, washed and stored at -80°C. Serum was isolated from an additional 10 ml of unheparinized whole blood and stored at -80°C.
Genomic DNA was isolated from blood lymphocytes, and GSTM1 genotyping (null versus non-null) was performed according to the method as described by Bell et al. (16). A multiplex PCR protocol was used to analyze for the presence or absence of GSTT1 gene, using a modification of the method described by Arand et al. (17). All primers (GSTT1 and albumin; primers for GSTM1 were not included) were at final concentrations of 50 pmol per 30 ml PCR reaction. The polymerase was rTaq (Pharmacia, Piscataway, NJ, USA), and dimethylsulphoxide (5%) was included. The annealing temperature was 55°C. 36 cycles were used. Products were resolved and visualized on 4% Nusieve gels (FMC Bioproducts, Rockland, ME). No attempt was made to distinguish homozygous from heterozygous individuals for the presence of the gene. GSTP1 genotyping was performed according to the method described in Harries et al. (18). Informative GSTM1, GSTT1, and GSTP1 genotypes were obtained in 159 (70%), 157 (69%) and 159 (70%) case patients and 164 (74%), 162 (73%) and 163 (73%) control subjects, respectively.
NAT1 genotyping was conducted as described previously for several published sequence variations in the 3' region of NAT1 near the putative polyadenylation signal; namely, NAT1*10 (T1088A, C1095A), NAT1*3 (C1095A), and NAT1*11 (9 bp deletion between 10651090) (19). Informative NAT1 genotypes were obtained in 156 (68%) cases and 163 (73%) control subjects. Because the NAT1*10 allele, containing an altered polyadenylation signal, has been associated with elevated levels of DNA adducts and higher risk of bladder cancer (13,20), genotypes containing at least one NAT1*10 allele were compared with other, non-NAT1*10 genotypes (NAT1*10 versus non-NAT1*10 genotypes). The rare non-functional NAT1 alleles (21) and other single nucleotide polymorphisms in NAT1 were not analyzed in this study.
Urine sample collection and cytochrome P4501A2 and NAT2 phenotyping
We obtained urine samples from 159 (70%) case patients and 144 (65%) control subjects. CYP1A2 and NAT2 phenotypes were determined in all of the case patients and in 142 of the control subjects. Each subject was given two packets of instant coffee (about 70 mg of caffeine) to be drunk between 3 and 6 p.m. The subject collected an overnight urine sample (ending with the first morning void) into a 1 litre plastic bottle that was picked up and processed the same day. On the day of collection, the subject was briefly interviewed about caffeine intake (in addition to the prescribed packets of instant coffee) and use of acetaminophen on the previous day. (Excessive caffeine consumption (>300 mg or >4 cups of coffee) has been shown to affect the validity of the phenotyping assay, and acetaminophen is used as the internal standard for the methylxanthine and urate assays so the presence of this compound may affect assay validity (22).) The urine specimens were acidified (400 mg of ascorbic acid per 20 ml of urine) prior to storage at -20°C (23). CYP1A2 phenotype determinations were performed according to the method of Kalow and Tang (22). Higher values of the CYP1A2 index reflect higher CYP1A2 activities. The NAT2 acetylator phenotype (slow, rapid) was determined using methods described previously (23).
Statistical analysis
Data were first analyzed by standard matched-pair methods including conditional logistic regression (24). The associations of bladder cancer with various exposure indices were measured by odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). When the case patient or the matched control subject failed to answer the relevant questions, we eliminated that casecontrol pair from the corresponding analysis. In addition, we broke the casecontrol matching and used unconditional logistic regression methods to analyze data on all informative case patients and control subjects (24). Results were similar in the matched and unmatched analyses. We present results from unconditional logistic regression in order to maximize the number of individuals included in the analyses.
Data presented in this report are derived from analyses with adjustment for cigarette smoking, an established risk factor in bladder cancer etiology (25). We repeated all analyses with and without further adjustment for other risk or protective factors for bladder cancer identified in this study, including use of non-steroidal anti-inflammatory drugs (26), high-risk occupations (truck/bus/taxi driver, aluminum product worker, hairdresser) (1), and estimated dietary carotenoid intake (unpublished data). There were no material changes in the results with or without adjustment for these potential confounders. The presented results do not include adjustment for these non-smoking-related risk factors.
We developed an algorithm for classifying study subjects into CYP1A2-slow and CYP1A2-rapid individuals: The mean value for CYP1A2 index among the 64 subjects who smoked around the time of urine collection was 8.18, while the comparable value among the 228 non-smokers was 4.75. Therefore, we first stratified study subjects by their smoking status around the time of urine collection (smokers, non-smokers). Among non-smokers, subjects with CYP1A2 index levels below the subgroup median were labeled as CYP1A2-slow while those with above median values were classified as CYP1A2-rapid. Among smokers, the range of smoking intensity (i.e. number of cigarettes smoked per day) was relatively narrow and the slope of the regression line between CYP1A2 index and number of cigarettes smoked per day around the time of urine collection was not statistically different from zero (2-sided P value = 0.40). Therefore, among smokers, we did not further adjust the value of CYP1A2 index by the number of cigarettes smoked per day. Smokers whose levels were above the subgroup median were labeled as CYP1A2-rapid while those with below median values were classified as CYP1A2-slow.
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Results |
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Table II shows the results of stratification by CYP1A2 phenotype on the hair dyecancer relationship. Irrespective of the exposure index under examination, CYP1A2 slow phenotype individuals displayed a stronger hair dyebladder cancer association compared with individuals with the rapid CYP1A2 phenotype. In fact, statistically significant associations between permanent hair dye use and bladder cancer risk were observed only among subjects exhibiting the slow CYP1A2 phenotype (P for trend = 0.01, 0.01, and 0.03, for duration of use, frequency of use and cumulative lifetime use, respectively).
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Discussion |
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In the present study, we also found that bladder cancer risk associated with permanent hair dye use is mainly confined to individuals with slow (versus rapid) CYP1A2 phenotype. Although N-oxidation by hepatic CYP1A2 is an established activation pathway of carcinogenic arylamines, hepatic ring-oxidation is generally regarded as a critical detoxification pathway (36). Arylamine ring-oxidation generally results in the formation of phenolic derivatives, which are efficiently conjugated and excreted. It has been shown that the carcinogenicity of 4-substituted 1,3-phenylenediamines (arylamines used in dyes) is reduced substantially or eliminated completely by oxidation of one or both amine groups or by N-substitution (30). Although N-oxidation and ring-oxidation are carried out by several cytochrome P450 monooxygenases, liver CYP1A2 possesses the highest catalytic activity (3,5,31).
Aromatic amines in hair dyes are absorbed through the scalp; thus, skin should be considered an important organ (main organ of entry) in the hair dyebladder cancer relationship. Skin is not only a mechanical barrier to the chemical ingredients of hair dyes, but also possesses the ability to detoxify carcinogenic arylamines (32). It has been shown that human skin and keratinocytes express the NAT1 enzyme (8,9), and that N-acetylation (usually a detoxification pathway) of aromatic amines in the skin is attributable to the polymorphic NAT1 (8). Thus, individuals with NAT1*10 genotypes, which in some reports has been associated with higher levels of gene expression, might detoxify aromatic amines in the skin to a greater extent that non-NAT1*10 acetylators (33).
Paraphenylenediamine (PPD), an arylamine, is a principal ingredient of hair dyes in the market today (34). It is the most common active ingredient and the most widely used primary intermediate in hair dye formulations (35). Hair dye users are exposed to PPD through the skin and N-acetylation of PPD in skin, which is catalyzed mainly through NAT1 enzyme, is suggested to be a detoxification pathway (8). PPD is acetylated to monoacetyl-PPD (MAPPD), which, in turn, is acetylated to N,N'-diacetyl-PPD (DAPPD), both by NAT1 activity. Moreover, formation of MAPPD or DAPPD is competitively inhibited in the presence of p-aminobenzoic acid, a NAT1-specific substrate, but not by sulfamethazine, a substrate of NAT2. These kinetic characteristics suggest that N-acetylation of PPD in human skin is predominantly attributable to the polymorphic NAT1 (8). Thus, if NAT1*10 genotype does affect NAT1 expression levels, these individuals may have increased detoxification of PPD in the skin, and may be relatively protected from hair dye exposure.
The role of hereditary polymorphisms of the arylamine NAT1 gene in the etiology of urinary bladder cancer is controversial. NAT1*10 genotype was recently reported as a general protective factor in bladder cancer in a German study (29). NAT1*10 was strongly protective among women, non-smokers, and among those never employed in at-risk occupations. Consistent with these findings, the present study noted a modifying effect of NAT1 genotype on the permanent hair dyebladder cancer association among lifelong non-smoking women. This modifying effect of NAT1 genotype persisted after adjustment for NAT2 phenotype. It may not be surprising that the modifying effect of the NAT1 genotype is absent among smokers. One explanation is that the protective effect of NAT1*10 is masked in smokers who are exposed chronically to 4-aminobiphenyl (4-ABP), an aromatic amine present in cigarette smoke, which can down-regulate NAT1 in skin. In fact, recent data suggest that the hamster or rat genes that are homologous to human NAT1 are down-regulated following 4-ABP exposure (28). An additional explanation for the lack of modification of the NAT1 genotype on the hair dyecancer association among smokers may be attributable to the potential dual role of NAT1 in extrahepatic tissues catalyzing O-acetylation (an activation pathway) of metabolites of arylamines such as those in tobacco smoke in bladder cells (7,13), while catalyzing N-acetylation (a detoxification pathway) of PPD contained in hair dyes in skin (8). Thus, a user of permanent hair dyes who also smoked would experience opposing effects from the two distinct sources of arylamine. An individual exhibiting the non-NAT1*10 genotype, which has been associated with lower levels of gene expression, would be relatively protected from smoking exposure but would experience an elevated risk from hair dye exposure. Conversely, an individual exhibiting the NAT1*10 genotype, which has been associated with higher levels of gene expression, would be relatively protected from hair dye exposure but would experience a higher proximal dose of arylamine to the target cells (i.e. bladder) from cigarette smoking. In other words, among smokers, the predicted modification effect of NAT1 genotype on the hair dyecancer relationship would be masked by the genes opposing effect on the smoking-cancer association.
In addition to cytochrome P450 isoenzymes, aromatic amines can be metabolically activated by various peroxidases, including cyclooxygenase 1 and 2 enzymes (COX1 and 2) (36), which are present in human bladder epithelium (3739). It is not known if PPD, a major aromatic amine present in hair dye solutions, is a substrate for COX1 and 2. However, there is some evidence that another phenylenediamine that used to be present in hair dye solutions, namely, 4-methoxy-m-phenylenediamine, is a known substrate for the COXs (4042).
At least eight other studies have provided some information on personal use of hair dyes and bladder cancer risks (4350). Six out of the eight studies investigated only general use of hair dyes (regardless of type) and bladder cancer risk (4348). One of these six studies was the largest casecontrol study of bladder cancer ever conducted: a multicenter, population-based study coordinated by the US National Cancer Institute (43). That study did not observe an overall association between hair dye use and bladder cancer risk (RR = 0.9 in women and 1.1 in men). However, an elevated relative risk was observed for users of black colored hair dye (RR = 1.4, 95% CI = 1.0, 1.9). The other five studies involved relatively small numbers of exposed subjects (4448). In two of the five studies, the overall estimates were around 1.5 but not statistically significant (46,47). In the Canadian study (45), no effect was seen in women (OR = 0.7, 95% CI = 95% CI = 0.3, 1.4), but among men, eight patients and no controls reported use of hair dye (1-sided P = 0.004). The case-control study from New Orleans (48) found an increased risk of bladder cancer for the number of times that hair dye was used per year among white women (P = 0.05). Only two studies have specifically examined permanent hair dyes in relation to bladder cancer (non-permanent dyes were not investigated in either study) (49,50). The first was a prospective cohort investigation involving 37 cases of urinary tract cancer, yielding a relative risk estimate of 0.6, based on five cases of urinary tract cancer versus 7.4 expected (49). The second study was the American Cancer Society Cancer Prevention Study II cohort investigation, which examined only deaths, not incident cases (50). No association was observed between use of permanent hair dyes and death from bladder cancer (RR = 1.08, 95% CI = 0.84, 1.38). It should be noted that fatal cases of bladder cancer constitute only 20% of incident cases of bladder cancer in the US (51).
In conclusion, we have provided additional evidence in support of a causal association between permanent hair dye use and bladder cancer risk. Our results implicate arylamines contained in hair dye solutions as the putative carcinogenic substances responsible for bladder cancer development in users of permanent hair dyes, by demonstrating that differences in arylamine activation and detoxification pathways substantially modify the overall relationship.
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Notes |
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Acknowledgments |
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References |
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