Interindividual variation in CYP1A1 expression in breast tissue and the role of genetic polymorphism

Regine Goth-Goldstein1,4, Martha R. Stampfer2, Christine A. Erdmann3 and Marion Russell1

Lawrence Berkeley National Laboratory,
1 Environmental Energy Technology Division,
2 Life Sciences Division and
3 Information and Computing Sciences Division, One Cyclotron Road, Berkeley, CA 94720, USA


    Abstract
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 Abstract
 Introduction
 References
 
The cytochrome P4501A1 (CYP1A1) enzyme is regulated at the transcriptional level and its expression is influenced by genetic factors, polymorphisms in the structural and regulatory genes, and by environmental factors such as exposure to polycyclic aromatic hydrocarbons (PAHs). To investigate the role of CYP1A1 in breast cancer, we studied CYP1A1 expression in breast tissue, thereby taking all possible modifying factors into account. We measured CYP1A1 expression in 58 non-tumor breast tissue specimens from both breast cancer patients (n = 26) and cancer-free individuals (n = 32) using a newly developed reverse transcription–polymerase chain reaction assay. CYP1A1 expression varied between specimens ~400-fold and was independent of age. CYP1A1 expression was somewhat higher in tissue from breast cancer patients than in that from cancer-free individuals, but this difference was not statistically significant. Analysis for CYP1A1 genetic polymorphisms revealed eight variants, seven in the cancer-free group and one in the patient group. The variant genotype was not a good predictor of expression level. We conclude that high CYP1A1 expression could be a risk factor for breast cancer and that the known CYP1A1 polymorphisms are not good predictors of CYP1A1 expression.

Abbreviations: CYP1A1, cytochrome P4501A1; PAHs, polycyclic aromatic hydrocarbons.


    Introduction
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 Abstract
 Introduction
 References
 
Polycyclic aromatic hydrocarbons (PAHs), a class of chemicals that includes potent carcinogens, could have a role in breast cancer because they accumulate in breast adipose tissue (1) and because normal human mammary cells in culture activate PAHs efficiently (2). PAH–DNA adduct levels have been found to be significantly higher in normal breast tissue of breast cancer patients than in that of non-cancer controls (3). The mutational spectrum in the p53 gene in breast tumors resembles that of lung cancers where there is a well-established role for environmental agents, such as tobacco smoke (4). The major metabolic pathway for ingested or inhaled PAHs to water-soluble derivatives is oxidative activation by CYP1A1 followed by detoxification by phase II enzymes. There is evidence supporting a role of CYP1A1 in breast cancer from recent animal experiments: using a rat model to identify loci that control breast cancer susceptibility, one of the four loci mapped to CYP1A1 or a nearby locus (5).

Interindividual variation in carcinogen metabolism has been recognized as a determinant of susceptibility to various cancers (6). Genetic polymorphism is one potential source of variation. For CYP1A1, four genetic polymorphisms consisting of single base changes have been described (7); two of them have been studied extensively as genetic biomarkers of susceptibility to various cancers (6), including breast cancer (8). The first described variant, CYP1A1*2, is located in the 3' non-coding region of the CYP1A1 gene and introduces an MspI restriction endonuclease site (9,10). The second variant, CYP1A1*3, is strictly linked to CYP1A1*2 (7) and consists of an A->G transition in exon 7 that results in an amino acid substitution of Val462 to Ile462 (11). Several studies have suggested that this genotype increases susceptibility to various cancers, but the biochemical basis is unclear. It has been assumed that the CYP1A1*2 and CYP1A1*3 alleles lead to higher inducibility. Expression of CYP1A1 is regulated by the aryl hydrocarbon receptor, together with several other regulatory proteins. Increased transcription of the CYP1A1 gene reflects induction of the enzyme (12). CYP1A1 expression can be induced by exposure to PAHs and organochlorines (13). Besides environmental factors, genetic factors can modify CYP1A1 expression; these include the genotype of the structural gene and the genotype of regulatory genes, including the aryl hydrocarbon receptor. Therefore determining the amount of transcript or the actual level of the enzyme captures the influence of all potentially modifying factors and is a more sensitive tool than the analysis of the genotype of a single gene.

We have examined CYP1A1 expression as a possible breast cancer risk factor by comparing CYP1A1 expression in non-tumor breast tissue from 27 breast cancer cases and 32 cancer-free individuals. Although we did not measure CYP1A1 protein levels or CYP1A1 enzyme activity, mRNA levels and enzyme activities are known to be closely related (14,15). The case specimens were derived from 22 mastectomies (peripheral non-tumor tissue) and five contralateral to carcinomatous breast. The control specimens were obtained from 32 reduction mammoplasties. Tissue specimens were dissected and isolated from adipose and connective tissue, so that only epithelial material was stored frozen as organoids (16). The pathological diagnosis of the excised tumors was intraductal carcinomas for two cases and infiltrating ductal carcinoma for the other 20 cases. In two of the 22 cases, metastasis to axillary lymph nodes was observed, indicating more advanced disease. Samples were collected without respect to age and race. Only the age and disease status of the specimen donors are known. No information is available on donors' race, lifestyle, smoking habits or other potential confounding factors. Individuals undergoing reduction mammoplasty ranged in age from 15 to 68 years, and mastectomy patients ranged in age from 30 to 87 years.

To determine CYP1A1 transcript levels, we developed a reverse transcription–polymerase chain reaction (RT–PCR) assay that determines CYP1A1 expression relative to the constantly expressed ß-actin gene, thus controlling for varying sample sizes and RNA yield. Previously published primers designed to span an intron (thus excluding amplification of any contaminating genomic DNA) were used and generated products of 320 bp for CYP1A1 and 273 bp for ß-actin (17,18). PCR conditions and cycle numbers were optimized separately for each target sequence to ensure that the reaction was in the linear phase of product accumulation. A five-fold serial dilution of cDNA was amplified in separate reactions for CYP1A1 and ß-actin. After amplification, the products were mixed together before electrophoresis on a 10% native polyacrylamide gel. The gel was stained with SYBR Gold nucleic acid stain and scanned on a Molecular Dynamics STORM 860 optical scanner. The fluorescent signal for each band was quantified using ImageQuant software (Figure 1Go). We found that this assay for CYP1A1 expression is sensitive, reproducible and has a broad dynamic range. CYP1A1 expression was measured in 59 non-tumor breast tissues from individuals with breast cancer (n = 27) and from cancer-free individuals (n = 32). Only one of the 59 samples did not have amplifiable RNA. CYP1A1 quantification was repeated in a blinded assay for 20% of samples. The correlation between the original measurements and the respective repeats was 0.9878, indicating that the assay is highly reproducible. In experiments with human mammary epithelial cells in culture, we found that the amount of ß-actin transcript was independent of benzo[a]pyrene exposure, whereas CYP1A1 transcript levels increase in proportion to the dose (data not shown). In the present study, ß-actin transcript levels in the 58 specimens could be evaluated from one of the first two dilutions of the cDNA. In contrast, the whole range of dilutions was needed to determine the CYP1A1 transcript levels in all specimens, indicating the large variations between individuals in CYP1A1 expression. The CYP1A1:ß-actin transcript ratio varied between the lowest value of 0.17 to the highest value of 70, a >400-fold range. As seen in Figure 2Go, individuals in the control group were younger than those in the case group, but CYP1A1 expression did not change with the age of the donors. The correlation coefficient for the CYP1A1:ß-actin transcript ratio and age was –0.0357 for cancer patients and 0.0434 for controls, constituting persuasive evidence that CYP1A1 level and age are not correlated. The lack of a correlation with age indicates that the reduction in estrogen levels experienced with menopause does not influence the CYP1A1 level, even though an interaction between the aryl hydrocarbon receptor and the estrogen receptor pathways has been observed in several systems (13).



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Fig. 1. Polyacrylamide gel of CYP1A1 and ß-actin PCR products for three specimens. The cDNA from each specimen was diluted serially five-fold and several of these dilutions were amplified for each specimen. Lane 1, molecular weight standard; lanes 2–6, specimen 86P peripheral to tumor; lanes 7–10, specimen 71C contralateral; lanes 11–13, 184 cells that were included in each reaction as control to test for interexperimental variation; lane 14, negative control.

 


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Fig. 2. CYP1A1:ß-actin ratio as function of age of specimen donors; {square} represent values of reduction mammoplasty controls and • represent values of breast cancer cases.

 
CYP1A1 expression, represented by the CYP1A1:ß-actin transcript ratio, differed between groups: The arithmetic mean of the CYP1A1:ß-actin ratio was 9.55 (SD = 14.66) in specimens from breast cancer patients and 6.31 (SD = 6.91) in specimens from cancer-free individuals. This difference was not statistically significant (in a two-tailed t-test, t was –1.11 and P 0.27) in the small sample studied. Comparing the distribution of CYP1A1:ß-actin values, a fairly log-normal distribution of values is seen for cases and controls (Figure 3Go). The geometric mean of the CYP1A1:ß-actin ratio was 3.70 (SD = 4.90) in cases and 3.15 (SD = 4.05) in controls.



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Fig. 3. Distribution of CYP1A1 expression levels among breast cancer cases and reduction mammoplasty controls. The upper two histograms show the untransformed data. The bottom two histograms show the log-transformed data.

 
The large variation between individuals in CYP1A1 expression might be explained by unmeasured environmental or lifestyle factors, such as smoking, which is known to induce CYP1A1 expression. CYP1A1 expression is increased in lung tissue of patients with tobacco-induced lung cancer (19). Others have reported variation in CYP1A1 expression in lung tissue (15,20,21), including a recent report that found that CYP1A1 expression in females was more than twice that in males (22).

The CYP1A1*2 and CYP1A1*3 alleles have been associated with a phenotype of high gene induction in response to PAHs (11). To investigate to what extent the CYP1A1 genotype modifies CYP1A1 expression, the CYP1A1 genotype of all specimens was determined using PCR/restriction fragment length polymorphism analysis according to published procedures (7). A total of eight CYP1A1*2 and CYP1A1*3 alleles in 58 samples were detected: three CYP1A1*2 heterozygotes, three CYP1A1*2/ CYP1A1*1 heterozygotes and two CYP1A1*2 homozygotes. The case group had only one CYP1A1*2/CYP1A1*1 heterozygote while the control group had seven variants. When all CYP1A1 values are ranked (Figure 4Go), the CYP1A1 variants are distributed between the lowest and highest expression values. All heterozygous variants and the one homozygous CYP1A1*2 variant have CYP1A1 values below the mean CYP1A1 values. Only one homozygous CYP1A1*2 variant was among the five specimens with the highest CYP1A1 expression values, indicating that the polymorphism has at most a minor role in determining CYP1A1 expression.



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Fig. 4. CYP1A1:ß-actin ratio ranked for all individuals. Open bars represent the CYP1A1*1 (wild-type) genotype. Solid bars represent CYP1A1 polymorphic variants of the following categories: (a) CYP1A1*2 heterozygotes; (b) CYP1A1*2/CYP1A1*1 heterozygotes; (c) CYP1A1*2 homozygotes. CYP1A1:ß-actin ratios are given in parentheses for the polymorphic variants. The origin of each tissue specimen is given below the bar, R, reduction mammoplasty; P, peripheral to carcinoma; C, contralateral.

 
The CYP1A1*2 variant is located in the non-coding region of the gene, suggesting that the CYP1A1*2 polymorphism alters the inducibility of CYP1A1. The CYP1A1*3 variant is located in exon 7, which codes for the heme-binding region. A change in amino acids in this region could possibly result in a change in enzyme activity. An earlier study reported a 50% higher enzyme activity (11). However, using purified human recombinant CYP1A1*1 and CYP1A1*2, a more recent study did not find different benzo[a]pyrene activation (23). Another study reported no difference in the kinetics of the CYP1A1 polymorphic variants (24). Therefore, any change in CYP1A1 level in CYP1A1*3 seems to be the result of strict linkage to CYP1A1*2 polymorphism (7), which presumably alters the inducibility of the enzyme. Our data suggest that CYP1A1*2 polymorphism has a minor, if any, role in modifying CYP1A1 expression (Figure 4Go). If individuals with the CYP1A1 variant genotype were exposed to much lower levels of PAHs than individuals with the wild-type genotype, the impact of genotype on expression might be masked. In an earlier study, human mammary epithelial cells derived from 18 individuals were treated with benzo[a]pyrene and DNA adducts quantified (2). Among the strains examined were six derived from donors tested here for CYP1A1 expression and CYP1A1 genotype, including the two homozygous CYP1A1*2 and one of the heterozygous CYP1A1*2 variants identified here. Contrary to expectations, the two homozygous CYP1A1*2 alleles had the lowest amount of adducts, indicating that the CYP1A1*2 genotype did not increase DNA adduct formation. Besides activating xenobiotics, CYP1A1 also metabolizes 17 ß-estradiol to the less active 2-hydroxy estradiol (25). A recent study suggests that CYP1A1*2 may be a marker of altered estradiol metabolism and of increased susceptibility to estrogen-related breast cancer in African-Americans (26).

In conclusion, this study shows that breast tissue expresses a considerable range of CYP1A1 levels independent of age and genotype, reinforcing the importance of evaluating both genotype and phenotype. Although the results are not statistically significant in the small unselected specimen groups available, they suggest that increased PAH activation by CYP1A1 might play a role in initiation of breast cancer. Larger sample sizes will be required to corroborate these suggestive findings.


    Notes
 
4 To whom correspondence should be addressed E-mail: r_goth-goldstein{at}lbl.gov Back


    Acknowledgments
 
We thank Dr Paul Williams for help and advice with the statistical analysis. We also thank Dr Leeka Kheifits of EPRI for critical review of the manuscript. This research was supported by funds from the California Breast Cancer Research Program of the University of California, Grant Number 1RB-0429 (R.G.G.), by USAMRMC grant no. DAMD17-98-1-8062 (RGG) and by the National Cancer Institute, Grant Number CA-24844 (M.R.S.). The work was performed under US Department of Energy contract no. DE-AC03-76SF00098.


    References
 Top
 Abstract
 Introduction
 References
 

  1. Martin,F.L., Carmichael,P.L., Crofton-Sleigh,C., Venitt,S., Phillips,D. and Grover,P.L. (1996) Genotoxicity of human mammary lipids. Cancer Res., 56, 5342–5346.[Abstract]
  2. Bartley,J. and Stampfer,M. (1985) Factors influencing benzo(a)pyrene metabolism in human mammary epithelial cells in culture. Carcinogenesis, 6, 1017–1022.[Abstract]
  3. Li,D., Wang,M., Ghingra,K. and Hittelman,W.N. (1996) Aromatic DNA adducts in adjacent tissues of breast cancer patients: clues to breast cancer etiology. Cancer Res., 56, 287–293.[Abstract]
  4. Biggs,P., Warren,W., Venitt,S. and Stratton,M. (1993) Does a genotoxic carcinogen contribute to human breast cancer? The value of mutational spectra in unravelling the aetiology of cancer. Mutagenesis, 8, 275–283.[Abstract]
  5. Shepel,L.A., Lan,H., Haag,J.D., Brasic,G.M., Gheen,M.E., Simon,J.S., Hoff,P., Newton,M.A. and Gould,M.N. (1998) Genetic identification of multiple loci that control breast cancer susceptibility in the rat. Genetics, 149, 289–299.[Abstract/Free Full Text]
  6. Hirvonen,A. (1999) Polymorphisms of xenobiotic-metabolizing enzymes and susceptibility to cancer. Environ. Health Perspect., 107, 37–47.
  7. Cascorbi,I., Brockmoller,J. and Roots,I. (1996) A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Res., 56, 4965–4969.[Abstract]
  8. Dunning,A.M., Healey,C.S., Pharoah,P.D., Teare,M.D., Ponder,B.A. and Easton,D. (1999) A systematic review of genetic polymorphisms and breast cancer risk. Cancer Epidemiol. Biomarkers Prev., 8, 843–854.[Abstract/Free Full Text]
  9. Kawajiri,K. Nackachi,K., Imai,K., Yoshii,A., Shinoda,N. and Watanabe,J. (1990) Identification of genetically high risk individuals to lung cancer by polymorphisms of the cytochrome P4501A1 gene. FEBS Lett., 263, 131–133.[ISI][Medline]
  10. Garte,S. and Crosti,F. (1999) A nomenclature system for metabolic gene polymorphisms. In Vineis,P., Malats,N., Lang,M., d'Errico,A., Caporaso,N., Cuzick,J. and Boffetta, P. (eds) Metabolic Polymorphisms and Susceptibility to Cancer. Scientific Publication no. 148, International Agency for Research on Cancer, Lyon, France, 5–12.
  11. Hayashi,S.I., Watanabe,J., Nackachi,K. and Kawajiri,K. (1991) Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P4501A1 gene. J. Biochem., 110, 407–411.[Abstract]
  12. Whitlock,J.P. Jr (1999) Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol., 39, 103–125.[ISI][Medline]
  13. Safe,S.H. (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol. Ther., 67, 247–281.[ISI][Medline]
  14. Vanden Heuvel,J.P., Clark,G.C., Kohn,M.C., Tritscher,A.M., Greenlee,W.F., Lucier,G.W. and Bell,D.A. (1994) Dioxin-responsive genes: examination of dose–response relationships using quantitative reverse transcriptase–polymerase chain reaction. Cancer Res., 54, 62–68.[Abstract]
  15. Willey,J.C., Coy,E.L., Frampton,M.W., Torres,A., Apostolakos,M.J.. Hoehn,G., Schuermann,W.H., Thilly,W.G., Olson,D.E. and Hammersley,J.R. (1997) Quantitative RT–PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. Am. J. Resp. Cell Mol. Biol., 17, 114–124.[Abstract/Free Full Text]
  16. Stampfer,M.R. (1985) Isolation and growth of human mammary epithelial cells. J. Tissue Culture Methods, 9, 107–116.
  17. Hayashi,S., Watanabe,J., Nakachi,K., Eguchi,H., Gotoh,O. and Kawajiri,K. (1994) Interindividual difference in expression of human Ah receptor and related P450 genes. Carcinogenesis, 15, 801–806.[Abstract]
  18. Horikoshi,T., Danenberg,K., Stadlbauer,T. et al. (1992) Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. Cancer Res., 52, 108–116.[Abstract]
  19. McLemore,T.L., Adelberg,S., Lui,M.C. et al. (1990) Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene regulation in primary pulmonary carcinomas. J. Natl Cancer Inst., 82, 1333–1339.[Abstract]
  20. Mace,K., Bowman,E.D., Vautravers,P., Shields,P.G., Harris,C.C. and Pfeifer,A.M. (1998) Characterisation of xenobiotic-metabolising enzyme expression in human bronchial mucosa and peripheral lung tissue. Eur. J. Cancer, 34, 914–920.[ISI][Medline]
  21. Raunio,H., Hakkola,J., Hukkanen,J., Lassila,A., Paivarinta,K., Pelkonen,O., Anttila,S., Piipari,R., Boobis,A. and Edwards,R.J. (1999) Expression of xenobiotic-metabolizing CYPs in human pulmonary tissue. Exp. Toxicol. Pathol., 51, 412–417.[ISI][Medline]
  22. Mollerup,S., Ryberg,D., Hewer,A., Phillips,D.H. and Haugen,A. (1999) Sex differences in lung CYP1A1 expression and DNA adduct levels among lung cancer patients. Cancer Res., 59, 3317–3320.[Abstract/Free Full Text]
  23. Zhang,Z.-Y., Fasco,M.J., Huang,L., Guengerich,F.P. and Kaminsky,L. (1996) Characterization of purified human recombinant cytochrome P4501A1-Ile462 and Val462: assessment of a role for the rare allele in carcinogenesis. Cancer Res., 56, 3926–3933.[Abstract]
  24. Perrson,I., Johansson,I. and Ingelman-Sundberg,M. (1997) In vitro kinetics of two human CYP1A1 variant enzymes suggested to be associated with interindividual differences in cancer susceptibility. Biochem. Biophys. Res. Commun., 231, 227–230.[ISI][Medline]
  25. Zhu,B.T. and Conney, A.H. (1998) Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis, 19, 1–27.[Abstract]
  26. Taioli,E., Bradlow,H.L., Garbers,S.V., Sepkovic,D.W., Osborne,M.P., Trachman,J., Ganguly,S. and Garte,S.J. (1999) Role of estradiol metabolism and CYP1A1 polymorphisms in breast cancer risk. Cancer Detect. Prevent., 23, 232–237.[ISI][Medline]
Received May 22, 2000; revised July 18, 2000; accepted July 27, 2000.