Polymorphisms in CYP1A1 and breast carcinoma risk in a population-based case–control study of Chinese women

Authors

  • Sonia M. Boyapati Ph.D.,

    1. Department of Medicine, Center for Health Services Research, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee
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  • Xiao Ou Shu M.D., Ph.D.,

    Corresponding author
    1. Department of Medicine, Center for Health Services Research, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee
    • Department of Medicine, Center for Health Services Research, Medical Center East, Room 6009, Nashville, TN 37232-8300
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    • Fax: (615) 936-1269

  • Yu-Tang Gao M.D.,

    1. Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China
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  • Qiuyin Cai M.D., Ph.D.,

    1. Department of Medicine, Center for Health Services Research, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee
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  • Fan Jin M.D.,

    1. Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China
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  • Wei Zheng M.D., Ph.D.

    1. Department of Medicine, Center for Health Services Research, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee
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Abstract

BACKGROUND

Cytochrome P450 1A1 (CYP1A1) is involved in the 2-hydroxylation of estrogen, the hormone that plays a critical role in the etiology of breast carcinoma.

METHODS

The authors evaluated common polymorphisms in the CYP1A1 gene in relation to breast carcinoma risk in a large population-based case–control study among Chinese women, the Shanghai Breast Cancer Study. Because the CYP1A1*3 and CYP1A1*4 alleles were not detected in the study population, analyses were performed for CYP1A1*2A (T→C transition in the 3′ noncoding region) and CYP1A1*2C (A→G transition in exon 7, resulting in a substitution of Val for Ile) in 1134 patients with breast carcinoma and 1227 controls.

RESULTS

The frequencies of the variant allele were 38.3% and 38.8% among cases and controls (P = 0.91), respectively, for the CYP1A1*2A polymorphism, and 23.1% and 24.8% (P = 0.26) for the CYP1A1*2C polymorphism. Homozygosity for both variant alleles in these 2 polymorphic sites (CYP1A1*2B) was associated with a borderline significant odds ratio (OR) of 0.71 (95% confidence interval [CI], 0.47–1.06). The reduced risk was more pronounced among postmenopausal women with long duration (> 30 yrs) of menstruation (OR = 0.43; 95% CI, 0.19–0.99) or among women with a low waist-to-hip ratio (OR = 0.52; 95% CI, 0.28–0.94).

CONCLUSIONS

Results from the current study suggest that homozygosity for the CYP1A1*2A and CYP1A1*2C alleles in the CYP1A1 gene may be associated with a reduced risk for breast carcinoma, particularly among lean women with long-term endogenous estrogen exposure. Cancer 2005. © 2005 American Cancer Society.

Cytochrome P450 1A1 (CYP1A1) is an important Phase I enzyme expressed in breast tissue. This enzyme is involved in the metabolism of estrogens and the mammary carcinogens, polycyclic aromatic hydrocarbons (PAH) and heterocyclic amines (HCA).1 CYP1A1 catalyzes the initial reaction of PAH and HCA to form reactive products. For estrogens, CYP1A1 catalyzes 2-hydroxylation to form catechol estrogens.2, 3 Although both 2-hydroxy and 4-hydroxy estrogens, if not detoxified, may generate reactive oxygen species through redox cycling, the former does not induce tumors in animal models.4, 5 Furthermore, 2-hydroxy estrogens do not retain estrogenic activity and 2-methoxy estrogens have been shown to have cancer inhibitory effects.6, 7 Therefore, it is believed that estrogen 2-hydroxylation is likely to be an inactivation pathway for potent estrogens. Given the role of CYP1A1 in the metabolism of estrogens and mammary carcinogens, it is conceivable that functional polymorphisms in the CYP1A1 gene may be related to breast carcinoma risk.

Four common polymorphisms have been reported for the CYP1A1 gene, including CYP1A1*2A, a T to C substitution located in the 3′ noncoding region; CYP1A1*2C, an amino acid substitution of isoleucine to valine in codon 462 in exon 7; CYP1A1*3, specific to African Americans, an MspI restriction fragment length polymorphism (RFLP); and CYP1A1*4, an amino acid substitution of threonine to asparagine in codon 461 adjacent to CYP1A1*2C. Epidemiologic studies investigating the association between polymorphisms in the CYP1A1 gene and breast carcinoma have been conflicting—whereas some have shown that CYP1A1 polymorphisms were significantly associated with breast carcinoma risk, others have indicated no association.8–20

The inconsistent findings from previous studies are, perhaps, not unexpected, given the dual role of CYP1A1 in mammary carcinogen activation and estrogen 2-hydroxylation. The direction of the CYP1A1/breast carcinoma association may depend on the underlying exposure to PAHs and HCAs as well as the level of endogenous estrogens. Levels of endogenous estrogens are correlated with measures of body size (i.e., body mass index [BMI], waist-to-hip ratio [WHR]).21 In the current analysis of Chinese women living in Shanghai, the underlying exposure to PAHs and HCAs is low because the majority of the population rarely smoke cigarettes or seldom consume well-done meat. The purpose of our study is to evaluate the association between polymorphisms in the CYP1A1 gene and breast carcinoma risk in conjunction with endogenous estrogen exposure in a Chinese population with low exposure to PAHs.

MATERIALS AND METHODS

Study Population

The Shanghai Breast Cancer Study is a population-based case–control study conducted among Chinese women in Shanghai. The study was designed to recruit all eligible cases with breast carcinoma who were 25 to 64 years of age and newly diagnosed with breast carcinoma between August 1996 and March 1998, as well as a representative random sample of controls from the general population. All cases and controls were permanent residents of urban Shanghai who had no previous history of cancer and were alive at the time of interview. Through a rapid case ascertainment system, supplemented by the population-based Shanghai Tumor Registry, 1602 eligible cases with breast carcinoma were identified during the study period, and in-person interviews were completed for 1459 (91%) of them. The major reasons for nonparticipation were refusal (109 cases [6.8%]), death before the interview (17 cases [1.1%]), and inability to locate (17 cases [1.1%]). Cancer diagnoses for all patients were confirmed by two senior study pathologists through review of tumor specimen slides.

Controls were randomly selected from the female general population and frequency matched to cases by age (5-yr intervals). The number of controls in each age-specific stratum was determined in advance according to the age distribution of the incident cases of breast carcinoma reported to the Shanghai Tumor Registry during the period from 1990 to 1993. The Shanghai Resident Registry, which keeps registry cards for all adult residents in urban Shanghai, was used to randomly select controls. For each age-predetermined control, a registry card identifying a potential control of the same 5-year age group was randomly selected. Only the women who lived at the address during the study period were considered to be eligible for the study. In-person interviews were completed for 1556 (90.3%) of the 1724 eligible controls identified. Reasons for nonparticipation included refusal (166 controls [9.6%]) and death or a previous cancer diagnosis (2 controls [0.1%]).

Data Collection

During an in-person interview, a structured questionnaire was used to elicit detailed information on demographic factors, menstrual and reproductive history, hormone use, dietary habits, previous disease history, physical activity, tobacco and alcohol use, weight, and family history of cancer. All participants were measured for their current weight, waist and hip circumferences, and sitting and standing height by trained study interviewers using a standard protocol.22 Among those who completed the in-person interviews, 10-mL blood samples were collected from 1193 (82%) cases and 1310 (84%) controls using ethylenediaminetetraacetic acid or heparin Vacutainer tubes. These samples were processed on the same day at the Shanghai Cancer Institute (Shanghai, China), and aliquots of buffy coats (leukocytes) for each participant were collected in 2-mL vials and stored at −70 °C.

Genotyping

Genomic DNA specimens for cases and controls were extracted from buffy coat fractions using the Puregene DNA Purification Kit (Gentra Systems, Minneapolis, MN) following the manufacturer's protocol. DNA concentration was measured by the PicoGreen dsDNA quantitation kit (Molecular Probes, Eugene, OR). Ten nanograms of genomic DNA were used for each polymerase chain reaction (PCR).

The CYP1A1 genotypes at the CYP1A1*2A, CYP1A1*2C, and CYP1A1*4 sites were determined using a PCR-RFLP–based assay described previously.23 These assays were conducted in 2001 at the Molecular Epidemiology Laboratory at the Vanderbilt-Ingram Cancer Center (Nashville, TN). The primers for the CYP1A1*2A site were 5′-CAGTGAAGAGGTGTAGCCGCT-3′ and 5′-TAGGAGTCTTGTCTCATGCCT-3′. The same set of primers was used for the CYP1A1*2C and CYP1A1*4 sites: 5′-CTGTCTCCCTCTGGTTACAGGAAG-3′ and 5′-TTCCACCCGTTGCAGCAGGATAGCC-3′. The PCR reactions were performed in a PTC-200 Peltier thermal cycler (MJ Research Inc., Waltham, MA). Each 25 μL of PCR mixture contained 10 ng DNA, 1 × PCR buffer with 1.5 mM MgCl2 (Qiagen, Santa Clarita, CA), 0.2 mM each of dNTP, 0.8 μM of each primer, and 1.0 U of Qiagen HotStartTaq DNA polymerase. The reaction mixture was initially denatured at 95°C for 15 minutes, followed by 35 cycles of 94 °C for 30 seconds, 62 °C for 30 seconds, and 72 °C for 30 seconds. The PCR was completed by a final extension cycle at 72 °C for 7 minutes.

The restriction enzyme MspI was used to distinguish the CYP1A1*2A polymorphic allele. The polymorphic allele (CYP1A1*2A) creates a new MspI restriction site. The PCR product (340 base pair [bp]) with the CYP1A1*2A allele was digested to 2 fragments (200 bp and 140 bp), whereas the PCR product with the wild-type allele remained undigested (340 bp). The restriction enzyme BsrDI was used to distinguish the CYP1A1*2C polymorphic allele. The PCR product (204 bp) with the wild-type allele was digested to 2 fragments (149 bp and 55 bp), whereas the PCR product with the CYP1A1*2C allele remained undigested (204 bp). The restriction enzyme BsaI was used to distinguish the CYP1A1*4 polymorphic allele. The PCR product (204 bp) with the wild-type allele was digested to 2 fragments (139 bp and 65 bp), whereas the PCR product with the CYP1A1*4 allele remained undigested (204 bp). The restricted products were analyzed by electrophoresis in 2% agarose gel containing ethidium bromide. The allele presenting none of the CYP1A1*2A and CYP1A1*2C was designated as CYP1A1*1A. The allele presenting both CYP1A1*2A and CYP1A1*2C was designated as CYP1A1*2B.

The laboratory staff was blind to the identity of the subjects. Quality control (QC) samples were included in the genotyping assays. Each 96-well plate contained 1 water, 2 CEPH 1347-02 DNA, 2 blinded QC DNA, and 2 unblinded QC DNA samples. The blinded and unblinded QC samples were taken from samples of randomly selected participants included in the study. Genotyping data were available for 1120 cases/1196 controls for CYP1A1*2A (a 94% and 91% success rate for cases and controls, respectively) and for 1131 cases/1209 controls for CYP1A1*2C (a 95% and 92% success rate for cases and controls, respectively). The major reasons for incomplete genotyping were insufficient DNA samples and unsuccessful PCR analysis.

Statistical Methods

Standard techniques for case–control studies were used. The odds ratio (OR) was the measure of association used to relate case–control status to genotype. For all ORs, 95% confidence intervals (CIs) were calculated. Cutoff points for BMI and WHR were 25 and 0.80, respectively, according to the usual definition of overweight (BMI) or median distribution among controls (WHR). Menopausal status was determined as cessation of menstruation for ≥ 12 months, excluding cessation of menses caused by pregnancy or breastfeeding. Menopausal age was calculated by subtracting the date of the last menstrual cycle from the date of birth. Years of menstruation was calculated by taking age at menopause (for postmenopausal cases and controls) or date of diagnosis (premenopausal cases) or date of interview (for premenopausal controls) and subtracting age at menarche as well as total months of pregnancy and breast-feeding. In the current analysis, median distribution among controls was used as the cutoff points for age at menopause and years of menstruation. Cases and controls with surgically induced menopause were excluded from the analyses involving menstruation.

Stratified analyses were conducted to explore the potential modifying effect of nongenetic risk factors in the CYP1A1-breast carcinoma association. The basis for the assessment of confounding factors included biologic plausibility, potential data-based confounders for this dataset, or risk factors known to be important to the Chinese population studied in this dataset, and whether inclusion of the variable in the model changed the risk estimate for the primary exposure variable of interest by ≥ 10%, either alone or in combination with other potential confounding variables. Potential confounders were age, education, income, total caloric intake, total meat intake, family history of breast carcinoma, history of fibroadenoma, age at menarche, parity, age at first live birth, months of breast-feeding, BMI, smoking status, and amount of exercise during the past 10 years. The final covariates that met these criteria were age, total energy intake, family history of breast carcinoma, income, education, and previous diagnosis of fibroadenoma. Unconditional multivariate logistic regression models were used to obtain final estimates. All statistical tests were based on two-tailed probability values.

RESULTS

Comparison of demographics, traditional breast carcinoma risk factors, and selected dietary factors by case and control groups are shown in Table 1. In general, cases were slightly older, more likely to have a family history of breast carcinoma among first-degree relatives, less likely to be physically active, had a higher BMI, and had a higher WHR compared with their control counterparts. Compared with controls, cases also had an earlier age at menarche, older age at menopause, and older age at first live birth. On average, cases had a higher daily caloric intake and consumed more fat when compared with their control counterparts. There were no significant differences in demographic characteristics between those who provided genotyping data and those who did not.

Table 1. Selected Characteristics of Study Population, Shanghai Breast Cancer Study, 1996–1998a
CharacteristicsBreast carcinoma cases (n = 1120) (%)Controls (n = 1196) (%)P value Breast carcinoma cases vs. controls
  • a

    Unless otherwise specified, means ± standard deviations are presented.

Demographics   
Age (yrs)47.6 ± 8.047.2 ± 8.80.24
Education   
 No formal education3.85.9 
 Elementary school8.48.6 
 Middle or high school75.975.6 
 College or higher11.910.00.08
Family history   
 First-degree relative with breastcarcinoma3.42.30.13
Lifestyle/body size   
 Physically active in the past 10 yrs19.425.6<0.01
 Body mass index (kg/m2)23.5 ± 3.40.03
 Waist-to-hip ratio0.81 ± 0.060.80 ± 0.06<0.01
 Regularly drink alcohol3.63.90.72
 Regular smoker2.52.40.91
 Ever used oral contraceptives21.421.30.95
Dietary intake   
 Total energy intake (kcal/day)1872 ± 4681847 ± 4580.18
 Total fat (gm)36.6 ± 17.835.3 ± 16.10.07
 Total fruit and vegetables (g/day)501 ± 276497 ± 2780.68
Reproductive factors   
 Age at menarche (yrs)14 ± 1.615 ± 1.7<0.01
 Duration of menstruation (yrs)31 ± 5.530 ± 5.9<0.01
Ever had a live birth94.895.90.21
 No. of live births1.4 ± 0.91.5 ± 0.90.10
 Age at first live birth (yrs)26.8 ± 4.126.2 ± 3.8< 0.01
 Months of breastfeeding12.0 ± 13.112.9 ± 14.60.11
Postmenopausal32.936.20.09
 Age at menopause (yrs)48.2 ± 4.747.5 ± 4.90.05

The distribution of the CYP1A1*2A and CYP1A1*2C genotypes is shown in Table 2. The CYP1A1*3 and CYP1A1*4 polymorphisms were not detected in this population and are thus not included in any analyses. Women who were homozygous for the CYP1A1*2C variant allele alone (OR = 0.75; 95% CI, 0.51–1.11) or were variant for both the CYP1A1*2A and CYP1A1*2C alleles (CYP1A1*2B) (OR = 0.71; 95% CI, 0.47–1.06) had a nonsignificantly reduced risk for breast carcinoma, and the reduction appeared to be slightly stronger among postmenopausal women (OR = 0.65; 95% CI, 0.33–1.31 and OR = 0.67; 95% CI, 0.34–1.32, respectively). Postmenopausal women with the CYP1A1*2A variant allele alone had risks that were slightly but nonsignificantly reduced.

Table 2. Multiple-Adjusted Associations between the CYP1A1 Genotype and Breast Carcinoma Risk, Shanghai Breast Cancer Study, 1996–1998
CharacteristicsAll womenPremenopausal womenPostmenopausal women
Cases/controlsbOR (95% CI)Cases/controlsOR (95% CI)Cases/controlsOR (95% CI)
  • OR: odds ratio; 95% CI: 95% confidence interval; ref: reference.

  • a

    For combined genotypes, information is missing for 17 cases and 49 controls.

  • b

    Adjusted for age, total energy intake, family history of breast carcinoma, income, education, and previous diagnosis of fibroadenoma.

  • c

    Combined CYP1A1*2A and CYP1A1*2C genotypes.

Genotypea      
 CYP1A1*2A      
 wt1/wt1433/4531.00 (ref)272/2771.00 (ref)147/1651.00 (ref)
 wt1/vt1517/5561.00 (0.72–1.21)350/3611.02 (0.81–1.28)157/1821.00 (0.73–1.37)
 Vt1/vt1170/1870.93 (0.72–1.21)113/1151.00 (0.73–1.38)55/700.86 (0.55–1.34)
CYP1A1*2C      
 wt2/wt2659/6941.00 (ref)213/2401.00 (ref)429/4411.00 (ref)
 wt2/vt2421/4421.07 (0.89–1.28)139/1551.07 (0.85–1.33)273/2731.09 (0.80–1.48)
 Vt2/vt251/730.75 (0.51–1.11)15/260.81 (0.50–1.31)34/440.65 (0.33–1.31)
Combined CYP1A1*2A and CYP1A1*2Cc      
 wt1/wt1, wt2/wt2 (CYP1A1*1A)429/4371.00 (ref)270/2661.00 (ref)145/1611.00 (ref)
 Wt1/vt1, wt2/wt2191/210 136/141 52/66 
 Wt1/wt1, wt2/vt24/60.87 (0.68–1.12)2/40.93 (0.69–1.24)2/20.88 (0.58–1.35)
  vt1/vt1, wt2/wt231/31 21/23 10/8 
  Wt1/vt1, wt2/vt2324/3381.01 (0.82–1.24)212/2120.97 (0.75–1.24)105/1161.02 (0.73–1.44)
  Wt1/wt1, vt2/vt20/0 0/0 0/0 
  Vt1/vt1, wt2/vt287/84 57/49 28/32 
  Wt1/vt1, vt2/vt20/10.96 (0.67–1.36)0/11.13 (0.74–1.72)0/00.95 (0.54–1.67)
  Vt1/vt1, vt2/vt2 (CYP1A1*2B)51/710.71 (0.47–1.06)34/430.74 (0.46–1.21)15/250.67 (0.34–1.32)

Exploratory analyses for the combined CYP1A1*2A and CYP1A1*2C genotypes and breast carcinoma risk stratified by indices of estrogen exposure are shown in Table 3. Significantly reduced risk of breast carcinoma was observed for women homozygous for both the CYP1A1*2A and CYP1A1*2C variant alleles (CYP1A1*2B) compared with those homozygous for the CYP1A1*2A and CYP1A1*2C wild-type alleles (CYP1A1*1A), especially among postmenopausal women with a longer duration of menstruation (OR = 0.43; 95% CI, 0.19–0.99), a lower BMI (OR = 0.45; 95% CI, 0.19–1.08 among postmenopausal women), or a lower WHR (OR = 0.52; 95% CI, 0.28–0.94).

Table 3. Multiple-Adjusted Associations between the CYP1A1 Genotype and Breast Carcinoma Risk, Stratified by Measures of Estrogen Exposure, Shanghai Breast Cancer Study, 1996–1998
VariablesaCYP1A1*2A and CYP1A1*2C wild-typeb (CYP1A1*1A)CYP1A1*2A variant/CYP1A1*2C wild-typeMixed CYP1A1*2A and CYP1A1*2CCYP1A1*2A variantc/CYP1A1*2C variant (CYP1A1*2B)
Cases/controlsOR (95% CI)Cases/controlsOR (95% CI)Cases/controlsOR (95% CI)Cases/controlsOR (95% CI)
  • WHR: waist-to-hip ratio; OR: odds ratio; 95%; CI: 95% confidence interval; BMI: body mass index.

  • a

    Allele combinations occur in a hypothesized order of increasing “at-risk” alleles.

  • b

    Adjusted for age, total energy intake, family history of breast carcinoma, income, education, and previous diagnosis of fibroadenoma.

  • c

    One control with three variant alleles included in this group.

  • d

    Among postmenopausal women.

  • e For combined genotypes, information is missing for 17 cases and 49 controls.

Years of menstruation        
 <30189/2111.00105/1300.99 (0.71–1.37)175/1971.07 (0.80–1.42)21/370.70 (0.39–1.24)
 >30187/1811.0094/950.97 (0.68–1.37)185/1791.08 (0.81–1.44)22/300.69 (0.38–1.25)
Years of menstruationd        
 304/71.001/43/41/2
 >30117/1331.0052/610.92 (0.59–1.44)108/1270.99 (0.69–1.41)10/220.43 (0.19–0.99)
BMI        
 <25310/3071.00149/1720.98 (0.77–1.26)275/3290.94 (0.77–1.14)37/520.80 (0.52–1.25)
 >25117/1301.0073/690.99 (0.68–1.44)140/991.54 (1.12–2.13)14/200.58 (0.28–1.20)
BMId        
 <2589/921.0034/480.74 (0.43–1.25)69/980.71 (0.46–1.09)8/190.45 (0.19–1.08)
 >2556/691.0028/261.32 (0.69–2.52)66/521.57 (0.95–2.61)7/61.46 (0.46–4.61)
WHR        
 <0.80179/2051.00106/1181.02 (0.73–1.42)191/2340.94 (0.71–1.23)18/390.52 (0.28–0.94)
 >0.80248/2321.00116/1230.88 (0.64–1.20)224/1941.08 (0.83–1.40)33/330.93 (0.55–1.56)
WHRd        
 <0.8054/581.0021/300.78 (0.40–1.52)42/620.82 (0.48–1.42)7/100.64 (0.21–1.90)
 >0.8091/1031.0041/441.00 (0.60–1.67)93/881.19 (0.79–1.78)8/150.60 (0.24–1.50)
Menopausal status        
 Premenopausal270/2661.00157/1641.03 (0.78–1.36)271/2651.11 (0.87–1.41)34/440.83 (0.51–1.35)
 Postmenopausal145/1611.0062/740.92 (0.61–1.37)135/1501.04 (0.75–1.43)15/250.62 (0.31–1.25)

In stratified analyses, we also examined whether factors related to PAH exposure (as measured by total red meat consumption, preference for deep-fried to well-done red meat, and passive smoke exposure) modified the association between the CYP1A1 genotype and breast carcinoma risk. In general, there were no explicit patterns of association across levels of factors related to PAH exposure (data not shown).

DISCUSSION

Our study found that homozygosity for both the CYP1A1*2A and CYP1A1*2C variant alleles (CYP1A1*2B variant allele) was associated with a reduced breast carcinoma risk, particularly among postmenopausal women with a long duration of estrogen exposure, women with a low WHR, or postmenopausal women with a low BMI. These findings appear to be consistent with the role of CYP1A1 in estrogen 2-hydroxylation.

Several epidemiologic studies have investigated the association between CYP1A1 polymorphisms and breast carcinoma risk, and some of them evaluated the potential modifying effect of CYP1A1 polymorphisms on the association of breast carcinoma with cigarette smoking and/or estrogen-related factors. No consistent pattern, however, has emerged from these studies. Four case–control studies investigated the effects of CYP1A1*2A in a Caucasian population and all of them reported no association.8–11 Of the two studies conducted among African Americans, one reported a positive association8 whereas the other reported no association with the CYP1A1*2A polymorphisms.9 One case–control study of Chinese women living in Taiwan, however, reported a significantly increased risk for breast carcinoma among those who were homozygous for the CYP1A1*2A allele.12 In the same population, after considering CYP1A1*2A together with polymorphisms in CYP17 and COMT, the risk for breast carcinoma was stronger in women with high numbers of high-risk genotypes (CYP17 A2/A2, CYP1A1*2A vt/vt, and COMT L/L) and either prolonged estrogen exposure or higher estrogen levels.13 In contrast to the Taiwanese study, a single case–control study of Japanese women reported a significantly decreased risk for breast carcinoma among those who were homozygous for the CYP1A1*2A variant allele compared with those with no CYP1A1*2A variant alleles.14 The sample size for both studies, however, was small.

Of eight case–control studies conducted in Caucasian populations that considered the CYP1A1*2C allele, all reported no overall association with CYP1A1*2C.8–11, 15–18 In a pooled analysis, after combining their data with 4 other published studies resulting in > 5000 total Caucasian subjects, Basham et al.18 reported no association between the CYP1A1*2C polymorphisms and breast carcinoma risk. However, in subgroup analyses, one group reported an increased risk with light smokers,16 another reported a suggested increased risk for early smoking,10 and a third reported an increased risk among women with serum polychlorinated biphenyls levels above the median of their control group.17 Two of the seven case–control studies also reported results separately for African Americans, with both studies reporting no association between CYP1A1*2C and breast carcinoma risk in this group.8, 9 In a small case–control study conducted among Chinese women in Taiwan, no association between CYP1A1*2C and breast carcinoma risk was reported.12 A recent Japanese study reported a significantly reduced risk for breast carcinoma among those with 2 variant CYP1A1*2C alleles,14 which is in line with the results from our study.

In Asians and Caucasians, the CYP1A1*2A and CYP1A1*2C polymorphisms are known to be in linkage disequilibrium.24 The CYP1A1*2A variant is located in the noncoding region of the gene, whereas the CYP1A1*2C variant is located in exon 7, which codes for the heme-binding region. It is conceivable that a change in amino acid in the heme-binding region could result in a change in enzyme activity. One study showed that the CYP1A1*2C polymorphism alters enzyme kinetics in generating diol metabolites from benzo[a]pyrene (B[a]P),25 another indicated that CYP1A1*2C results in a 3-fold increase in microsomal enzyme activity and mRNA levels in Asians,26 whereas other studies did not find any change in enzyme kinetics27 or B[a]p activation28 for CYP1A1*2C. In a small study, CYP1A1 mRNA was higher in Caucasians with the double heterozygous genotype for CYP1A1*2A and CYP1A1*2C, but not for CYP1A1*2C alone.20 Our study results appear to support a combined role of these two polymorphisms in breast carcinoma risk.

Significant ethnic differences have been observed in the distribution of the CYP1A1 polymorphic genotypes.29 Specifically, the CYP1A1*2A and CYP1A1*2C alleles occur much more frequently in Asians compared with Caucasians and African Americans. The distribution of genotypes in our Chinese population is similar to that seen in the Taiwanese and Japanese studies. In general, Asian women also have lower overall endogenous estrogen levels compared with their Western counterparts. We found that when combined CYP1A1*2A and CYP1A1*2C decrease the risk for breast carcinoma among Chinese women, particularly among postmenopausal women with lower BMI or women with a low WHR. A positive association between postmenopausal breast carcinoma and BMI has been reported in this population, as well as in a recent combined analysis of eight prospective studies.21, 22, 30 In the combined analysis, the increase in risk was substantially reduced by adjustment for serum estrogen concentration, suggesting that the increase in cancer risk with increasing BMI was largely associated with an increase in estrogen levels. If confirmed, our findings may aid in clarifying the mechanistic pathway involved in breast carcinoma reduction among Asian women with low endogenous estrogen levels.

The association of the CYP1A1 genotype with breast carcinoma may be modified by HCAs and PAHs, which are CYP1A1 substrates. However, our population consists of women who are essentially nonsmokers and rarely eat well-done meat. Additional analyses stratified by passive smoking exposure and red meat consumption/doneness index failed to find any noticeable change in the CYP1A1 polymorphism/breast carcinoma association (data not shown). Furthermore, other environmental PAH exposures (e.g. air pollution) are likely to have little variation in our study population of Chinese women living in Shanghai.

Our study found that breast carcinoma risk is significantly lower among postmenopausal women with a low BMI and a low WHR, indications of low endogenous estrogen exposure, and among women with increasing numbers of variant CYP1A1*2A and CYP1A1*2C alleles. CYP1A1 is involved in the 2-hydroxylation of estrogens. Although this is, in general, considered a detoxifying pathway, 2-hydroxy estrogens, if not conjugated, could enter redox cycling to generate reactive free radicals. Among women with high estrogen levels and high CYP1A1 activity, excess 2-hydroxy estrogens may overwhelm the detoxifying enzymes, thus negating the beneficial effect of high CYP1A1 activity. Conversely, according to this hypothesis, among women with low estrogen levels and high CYP1A1 activity, detoxification would lead to risk reduction. Our finding that the genotype associations were more pronounced among postmenopausal women with longer years of menstruation may not be completely contradictory to this hypothesis if women with longer years of menstruation also had low levels of endogenous estrogens. However, we were unable to evaluate that association in the current study. Although some of the positive findings might be due to chance, given the biologically plausibility of the overall results, more studies are needed to understand the joint effect of estrogen and the CYP1A1 genotype on breast carcinoma and to determine the exact mechanism involved.

Our study has several strengths. This is one of the largest studies to date to investigate the association between the CYP1A1 polymorphisms and breast carcinoma risk. In addition, our population of Chinese women is relatively homogenous. For example, > 98% of them are classified into a single ethnic group (Han Chinese), which minimizes potential bias due to population stratification. The high participation rate and population-based study design reduced potential selection bias. The extensive information on lifestyle factors allowed a comprehensive evaluation of their interaction or confounding effects on the association of genetic polymorphisms and breast carcinoma risk. Our study also has some limitations. After stratification, cell frequencies for many subgroup analyses were small, resulting in unstable estimates or possible chance findings. Therefore, we suggest these analyses to be exploratory and hypothesis generating, with the hope of stimulating future, larger-scale studies on this topic.

Results from the current study suggest that the CYP1A1*2A and CYP1A1*2C polymorphisms may contribute to lower breast carcinoma risk among Chinese women with low endogenous estrogen levels. Additional studies involving other ethnic groups are needed to confirm our findings.

Acknowledgements

The authors are grateful to the research staff for their help with the Shanghai Breast Cancer Study.

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