The results of our population-based case-control study are consistent with the hypothesis that both phase I and phase II metabolic enzymes may modify the relation between hair dye use and risk of NHL, although none of our findings achieved statistical significance after adjustment for multiple comparisons. Specifically, the effect modification by genetic variations in CYP2C9
, and GSTP1
genes in our study was mainly observed among women who started using hair dyes before 1980. However, the lack of findings among women who started using hair dyes in 1980 or later could have been due to lack of statistical power, as there were fewer subjects who started using hair dyes in 1980 or later (n
281) than before 1980 (n
Thus far, very few investigators have studied the relation between genetic polymorphisms in xenobiotic metabolic pathway genes and risk of NHL (14
). Results from the limited studies available have been inconsistent. One possible explanation for the contradictory results is that few investigators have considered environmental carcinogenic exposures when examining the gene-disease relation. Populations involved in different studies may have different levels of exposure to various substrates.
To date, only Morton et al. (2
) have examined genetic variation in NAT1
in relation to NHL risk and hair dye use. They found an increased risk of NHL associated with hair dye use among women who carried the NAT2
rapid/intermediate acetylator phenotype and started using hair dyes before 1980, but not among women who carried the slow NAT2
acetylator phenotype. In our study, we found a similarly increased risk of NHL associated with starting use before 1980 among women with rapid/intermediate NAT2
phenotypes versus slow NAT2
phenotypes, although a statistically significant increase was observed only for women with rapid/intermediate NAT2
phenotypes. NATs are involved in metabolism of a variety of aromatic and heterocyclic amines through N
-acetylation (detoxification) and/or O
-acetylation (activation) (20
rapid acetylators have greater O
-acetylation activity, resulting in increased bioactivation of aromatic and heterocyclic amines and formation of DNA adducts that have been shown to lead to cancer in rodents, including lymphoma (11
). As such, the observed higher risk associated with hair dye use for NAT2
rapid/intermediate acetylators is biologically plausible. However, the effect modification by NAT*10
genotypes observed in the previous study (2
) was not replicated in our study.
In addition to effect modification by NAT2
acetylator status, we observed effect modification by CYP
genes on the risk of NHL associated with hair dye use. CYP2C9
is involved in bioactivation of several carcinogens, such as polycyclic aromatic hydrocarbons and hetereocyclic aromatic amines, which are present in hair dyes (23
). The CYP2C9 Ex3-52C>T rs1799853
polymorphism has been shown to alter CYP2C9 activity (24
). As such, the finding of increased risk of NHL associated with hair dye use among women who carried the CYP2C9
variant allele, but not among those who carried 2 wild-type alleles, is biologically plausible. CYP2E1 is also a key enzyme that is involved in metabolic activation of a variety of carcinogenic and toxic compounds, including benzene and N
). Although the functional significance is currently unclear for the variant allele of CYP2E1 -332T>A rs2070673
, the finding from our study that women who carried the variant allele appeared to have higher risk of NHL associated with hair dye use, despite nonsignificant results for the gene-hair dye interaction term in the models, requires further investigation. Future functional studies on this single nucleotide polymorphism are necessary to explain the meaning of the observed modification effect of CYP2E1 -332T>A
plays a pivotal role in conjugation and detoxification of environmental carcinogens, such as arylamines (27
intron 6 rs1799735
, a 3-base-pair deletion polymorphism, produces a binding site for the transcription factor YY1 [Yin Yang 1], which influences the expression of GSTM3
). The GSTM3
3-base-pair deletion allele has been shown to alter expression in cytosol, which may result in varying efficiency of carcinogen detoxification and therefore may predispose a person to cancer (29
). This polymorphism has been reported to modify susceptibility to cigarette and tobacco carcinogens (30
). Our study revealed a modulation of risk associated with hair dye use by this polymorphism.
Our study also found that the GSTP1 Ex5-24A>G rs1695
polymorphism modified the relation between hair dye use and risk of NHL. This polymorphism results in an Ile105
Val alteration in the encoded amino acid sequence. It has been shown that the Val105
variant allozyme accommodates less bulky substrates than the Ile105
allozyme and subsequently displays different substrate specificities (34
). In addition, the Val105
allozyme exhibits different thermal stability than the Ile105
). These characteristics may be responsible for the apparent difference in the risk of NHL associated with hair dye use with respect to GSTP1
allozymes, although the P
value for interaction was not statistically significant.
In this study, we did not observe significant modulation of risk associated with hair dye use with respect to CYP1A1, CYP1A2, CYP1B1, GSTT1, or NAT1 genotypes. It is possible that the single nucleotide polymorphisms we studied for these genes are not functional single nucleotide polymorphisms. As such, a possible role of those genes cannot be ruled out.
Several strengths of this study lend confidence to the observed associations. All NHL cases were histologically confirmed by 2 study pathologists who are experienced in diagnosis of lymphoma, which minimized disease misclassification, particularly for NHL subtypes. Using a rapid case ascertainment system to identify newly diagnosed NHL cases reduced potential survival bias. Detailed information on lifetime hair dye use and potentially confounding variables allowed us to examine the effect of hair dye use by time period of use, with control for several potential confounders.
Although we had a relatively large sample, the numbers of subjects became small for stratified analyses by both hair dye use and genetic variation. Given the large number of tests conducted in the study, the possibility that some or all of the observed associations were due to chance cannot be ruled out. In this study, we included women only; therefore, the results may not be generalizable to men. Differential recall bias regarding hair dye use among study subjects could have occurred if NHL patients believed that hair dye use or use of a specific type (or color) of hair dye increased a person's risk of NHL. The lack of association between overall use of hair dye or hair-coloring products after 1980 and major NHL subtypes (e.g., diffuse large B-cell lymphoma) argues against recall bias’ having played a major role in the observed association. Additionally, although we were able to examine allelic/phenotypic classifications for NAT1 and NAT2, respectively, such detailed information is not known for the other metabolic genes studied here. Thus, we were limited to the examination of individual single nucleotide polymorphisms for the other genes, which may not have captured meaningful information on gene function.
In summary, the results from this study suggest effect modification of the relation between hair dye use (particularly use that started before 1980) and NHL by variation in several genes involved in xenobiotic metabolism: CYP2C9 Ex3-52C>T, CYP2E1 -332 T>A, the GSTM3 deletion in intron 6, GSTP1 Ex5-24A>G, and NAT2 acetylator status. Although there is biologic plausibility for the majority of these findings, the results should be replicated in other studies with larger samples before stronger conclusions can be made about the roles of these genes in the carcinogenicity of hair dye use.