CEBP Meeting Calendar Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cribb, A. E.
Right arrow Articles by Saleh, T. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cribb, A. E.
Right arrow Articles by Saleh, T. M.
Cancer Epidemiology Biomarkers & Prevention Vol. 15, 551-558, March 2006
© 2006 American Association for Cancer Research

Role of Polymorphic Human Cytochrome P450 Enzymes in Estrone Oxidation

Alastair E. Cribb1, M. Joy Knight1, Dagny Dryer2, Judy Guernsey3, Kimberly Hender2, Marvin Tesch2 and Tarek M. Saleh1

1 Laboratory of Comparative Pharmacogenetics, Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island; 2 Queen Elizabeth Hospital, Charlottetown, Prince Edward Island, Canada; and 3 Department of Community Health and Epidemiology, Dalhousie University, Halifax, Nova Scotia, Canada

Requests for reprints: Alastair E. Cribb, Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, Canada C1A 4P3. Phone: 902-566-0661; Fax: 902-566-0832. E-mail: acribb{at}upei.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen and its metabolites are believed to play important roles in breast cancer. The influence of genetic polymorphisms in the enzymes responsible for formation and disposition of estrogen on breast cancer risk may shed light on the importance of estrogen metabolites in this disease. However, for such studies to be valid, it is important to correctly identify the enzymes involved in estrogen bioactivation. Therefore, we assessed the human cytochrome P450–dependent oxidation of estrone using substrate concentrations that more closely approximate the maximum expected concentrations in breast tissue. The in vitro metabolism of estrone by recombinant human cytochrome P450 enzymes and human liver microsomes was studied. The formation of estrone metabolites (2-hydroxyestrone, 4-hydroxyestrone, and 16{alpha}-hydroxyestrone) was monitored by high-performance liquid chromatography. 2-Hydroxyestrone formation was catalyzed predominantly by CYP1A2, CYP1A1, and CYP1B1 enzymes; 4-hydroxyestrone formation was catalyzed predominantly by CYP1B1, CYP1A2, and CYP1A1 enzymes; and 16{alpha}-hydroxyestrone formation was catalyzed predominantly by CYP2C19, CYP1A1, and CYP3A5. This study confirms the important role of members of the CYP1 family in the 2-hydroxylation and 4-hydroxylation of estrone, but the enzymes identified as responsible for the 16{alpha}-hydroxylation of estrone are different from those previously identified. The relative importance of these enzymes in vivo would depend on the specific tissue expression of the enzymes. These enzymes are all known to be genetically variant in the human population, and additional studies to assess the role CYP1A2, CYP2C19, and CYP3A5 in breast cancer risk are indicated. (Cancer Epidemiol Biomarkers Prev 2006;15(3):551–8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Identification of endogenous and exogenous factors influencing breast cancer is necessary for a reliable assessment of risk and for designing strategies for controlling the disease. The endogenous estrogens (E) estradiol (E2) and estrone (E1) contribute to breast cancer risk through their role as growth promoters (1-4). Estrogen metabolites may also contribute to the initiation and promotion of breast cancer through the formation of DNA-damaging species or activation of the estrogen receptor (1, 4-7). Two major catechol metabolites, 4-hydroxy-estrogens (4-OH-Es) and 2-hydroxy-estrogens (2-OH-Es), have been implicated in breast cancer risk. Catecholestrogens are unstable metabolites that can lead to the generation of more reactive semiquinones and quinones and produce DNA-damaging reactive oxygen species (1, 5, 8-11). The 4-OH-Es are more unstable than the 2-OH-Es (12). The 4-OH-Es are equivalent in estrogenic potency but longer-acting than the parent estrogens because of a decreased dissociation from the estrogen receptor (13, 14). Although 2-OH-Es have some inherent estrogenic activity, they are considerably less potent than the parent estrogen and may therefore function as antiestrogens in vivo (14-18). Thus, 4-OH-Es are thought to increase breast cancer risk, whereas 2-OH-Es are thought to be protective.

Another major oxidative pathway is the formation of 16{alpha}-hydroxyestrogens (16{alpha}-OHE). 16{alpha}-OH-estrone is generally considered to be estrogenic and is thought to be associated with an increased risk of breast cancer (19, 20). 16{alpha}-OH-estrone has been shown to be genotoxic and cause aberrant proliferation of mammary cells (21, 22) and to covalently bind to the estrogen receptor (23).

The significance of these three estrogen metabolites in human breast cancer remains controversial. The major pathways for metabolism of estradiol are through conversion to estrone and oxidation to 2-OH-estrone and 16{alpha}-OH-estrone. Therefore, the majority of attention has focused on the formation of these metabolites. Many studies have shown an association between the urinary ratio of 2-OH-estrone to 16{alpha}-OH-estrone and breast cancer risk, but other studies have not found such an association (24-27). Another approach to understanding the significance of these metabolites in vivo has been to assess the influence of genetic polymorphisms in the enzymes responsible for their formation and disposition on breast cancer risk. In order for these studies to be interpreted, it is important to know the enzyme specificity of estrogen hydroxylation reactions.

Several studies have explored the specificity of these reactions, but the majority of studies were done at estrogen concentrations in the range of 50 to 100 µmol/L and often used prolonged incubation times to generate metabolites for quantification (28-37). Plasma concentrations of estrogen are generally very low, ranging between 0.4 and 70 nmol/L (38). Tissue concentrations of estrogens are not directly linked to circulating estrogen concentrations as a result of local synthesis, local degradation, and other factors (38-40). Tissue concentrations of estrogens in the breast are generally much higher than plasma concentrations, with estrone and estrone sulfate concentrations as high as 3 to 5 µmol/L in breast tissue or fluids (38, 41, 42). Furthermore, up to 80% of total estrogens found in breast tissue are in hydroxylated forms, suggesting local metabolism (39). The endogenous concentrations of estrone, whether circulating or in tissue, are considerably below those previously used to assess the specificity of CYP-mediated estrogen oxidation. Assessment of CYP specificity at high substrate concentrations, without adequate consideration of the expected in vivo concentrations, may result in erroneous identification of important enzymes. Therefore, we assessed the role of human cytochrome P450 enzymes in the oxidation of estrone at lower substrate concentrations and using shorter incubation times.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Reagents
Estrone, 2-OH-estrone, 4-OH-estrone, and 16{alpha}-OH-estrone were purchased from Steraloids, Inc. (Newport, RI). Chloramphenicol, furafylline, ketoconazole, omeprazole, sulfaphenazole, NADP+, reduced form (NADPH), and other routine chemicals were purchased from Sigma Chemical (St. Louis, MO). Ascorbic acid and high-performance liquid chromatography (HPLC)-grade acetonitrile were purchased from Fisher Scientific (Atlanta, GA). Perchloric acid was purchased from BDH Laboratory Supplies, VWR International (Missisauga, Ontario, Canada). N-3-benzyl-nirvanol was purchased from BD Biosciences Gentest Products and Services (Woburn, MA).

Microsomes and Recombinant Enzymes
This study was approved by the UPEI Ethics Committee. Pooled microsomes and single donor human liver microsomes were purchased from BD Biosciences Gentest Products and Services. Single donors included H006, HG23, HK27, HG30, HK31, HG42, HG43, HG56, HG89, HG93, HG103, HH2, HH9, HH31, HH48, and HH108. Microsomes were supplied at a protein concentration of 20 mg/mL in 250 mmol/L sucrose. The activities of known CYP substrates were provided by the supplier and were determined according to published protocols. Human recombinant baculovirus-expressed enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C18, CYP2C19, CYP2D6*1, CYP2E1, CYP2J2. CYP3A4, CYP3A4 + b5, CYP3A5, CYP3A7 + b5, CYP19, CYP4A11, and CYP4F12 were purchased from BD Biosciences Gentest Products and Services. Recombinant enzymes were suspended in 100 mmol/L Tris (pH 7.5), and the specific P450 content was provided by the supplier. Microsomes and recombinant enzymes were stored at –80°C until use.

Enzyme Assays
Microsomal incubations containing 1 mg/mL human liver microsomes, ascorbic acid (1 mmol/L), and NADPH (1 mmol/L) in PBS (pH 7.4; final volume of 250 µL) were preincubated in a shaking water at 37°C for 3 minutes. The reaction was then initiated by the addition of estrone at the specified concentrations. Estrone was prepared as a 100 mmol/L stock in DMSO and diluted so that the final concentration of solvent in the assays did not exceed 1%. The reaction was incubated in a shaking water bath for 10 minutes until terminated with the addition of 25 µL ice-cold 15% perchloric acid. The mixture was cooled on ice and then centrifuged for 10 minutes at 8,000 x g. The resulting supernatant was removed for HPLC analysis.

Recombinant enzyme assays were conducted in a similar fashion, except that the reaction was initiated with the addition of enzyme, rather than estrone, and the reactions were terminated after 5 minutes (or other time as specified). The concentration of recombinant enzymes used ranged from 10 to 100 pmol/mL and was adjusted as required to ensure linear formation of metabolites. Organic solvents can negatively affect the activity of cytochrome P450 enzymes (43-45). DMSO was routinely used as a solvent, but acetonitrile was employed for CYP2C19, CYP2C9, CYP3A4, and CYP3A5 to minimize effects on enzyme activity.

For inhibition studies, inhibitors were used at the concentration specified in the figures. All inhibitors were added before the addition of estrone. Furafylline was preincubated with the microsomes for 10 minutes at 37°C with NADPH before addition of estrone. Inhibition is expressed as the percent of the corresponding solvent control.

Immunoinhibition
Inhibitory monoclonal antibodies to the human cytochrome P450 1A2 (clone 26-7-5), 2C9 (clone 763-15-5), 2E1 (clone 1-73-18), 3A4/5 (clone 3-29-9), 2C19 (clone 1-7-4-8), and egg lysozyme (clone Hy-Hel-9; to serve as control) were generously provided by Dr. Harry Gelboin (National Cancer Institute, NIH, Rockville MD). Human-pooled microsomes (1 mg/mL), monoclonal antibodies (5, 10, or 20 µL), and PBS were preincubated for 5 minutes at 37°C in a shaking water bath. Ascorbic acid (1 mmol/L) and estrone (2.5 and 25 µmol/L in 0.5% ACN) were added and warmed in the shaking water bath for a further 2 minutes. The reaction was initiated with NADPH and incubated in the shaking water bath for 5 minutes. Control incubations were conducted using antibodies against egg lysozyme and antibodies not anticipated to inhibit estrone metabolism based on recombinant enzyme studies (e.g., CYP2E1 clone 1-73-18). The reaction was terminated with the addition of ice-cold 15% perchloric acid and centrifuged for 10 minutes at 8,000 x g. The resulting supernatant was removed for analysis by HPLC, as described below.

HPLC Analysis of Metabolites
Analysis of metabolites was done with an HPLC system (Shimadzu LC-10A series HPLC system equipped with two LC-10AT VP pumps, an SIL 10A autoinjector, an SCL-10A VP system controller, an RF-10AXL fluorescence detector, and Class-VP Chromatography Laboratory Automated Software; Shimadzu Scientific Instruments, Inc., 1998, Columbia, MD) with an Ultracarb 5-µm ODS (30) 4.6 x 150 mm analytic column (Phenomenex, Inc., Torrance, CA). Fifty microliters of the reaction supernatant was injected per run. Metabolites were separated with acetonitrile/water gradient at a flow rate of 1.25 mL/min with a linear gradient from 25% acetonitrile to 50% acetonitrile over 30 minutes. The column was then stabilized with 25% acetonitrile for 15 minutes before initiating the next run. Retention times for the estrogen standards were 16{alpha}-OH-estrone, 12.5 minutes; 2-OH-estrone, 19.0 minutes; 4-OH-estrone, 20.4 minutes; and estrone, 26.6 minutes. Metabolites were detected with a fluorescent detector with an excitation of 280 nmol/L and an emission of 445 nmol/L. Analytes were quantified against an external standard curve. Limits of quantitation were 2, 1, and 8 pmol, respectively, with coefficient of variations of 15% to 22% at the lowest concentration and <10% at higher concentrations.

Data Analysis
All incubations with recombinant enzymes were done in duplicate or triplicate, and experiments were repeated at least twice to confirm results. Experiments with >10% deviation between replicates were repeated to confirm results. As all incubations with recombinant enzymes were conducted with the same preparations, only the mean is reported. Km and Vmax values were derived from Hanes-Woolf plots. For experiments with human liver microsomes, a minimum of three individual livers (analyzed in duplicate or triplicate) were used and the results are presented as the mean ± SE. Correlations were assessed by Spearman's rank correlation coefficient. Effects of inhibitors were compared by one-way ANOVA to the corresponding vehicle control, with post hoc comparison with control by Bonferroni corrected t test. P < 0.05 was accepted as being statistically significant. All statistical analyses were done using GraphPad Prism version 3.03 (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial experiments determined that the linearity with time of estrone metabolism with recombinant enzymes and human liver microsomes was relatively short, regardless of whether NADPH or an NADPH-generating system was used. The linearity of product generation varied from 7.5 minutes up to 10 to 20 minutes, depending on the specific conditions (Fig. 1). Linearity of estrone disappearance was maintained with a loss of substrate of up to 20% at lower concentrations (2.5-5 µmol/L). Experiments with rat hepatic microsomes and recombinant human CYP enzymes revealed that NADPH-dependent metabolism of the metabolites occurred, causing a loss of metabolites with time (data not shown). This may contribute to loss of linearity.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Linearity of estrone metabolism with time in in vitro incubations. Linearity of 2-OHE1 formation from estrone by recombinant CYP2C9 (top; 5 µmol/L estrone, 1 mmol/L NADPH, 230 pmol/mL CYP2C9) or human liver microsomes (bottom; 10 µmol/L estrone, 1 mmol/L NADPH, 1 mg/mL human liver microsomes). CYP2C9 only formed 2-OH-estrone. Based on similar results with other recombinant enzymes and a variety of conditions with human liver microsomes, further reactions were restricted to 5 minutes with recombinant enzymes and a maximum of 10 minutes with human liver microsomes for inhibition studies.

 
To further assess the effect of prolonged incubation times on the results, kinetic studies were done with three recombinant enzymes and human liver microsomes using incubation times of 5 and 30 minutes (Table 1). The use of extended incubation times resulted in higher apparent Km values and, in some cases, reduced Vmax values. Therefore, to work as close as possible to initial rate conditions for estimation of kinetic values, incubation times were kept to 5 minutes for recombinant enzymes and to 10 minutes when performing studies with human liver microsomes.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of prolonged incubation times on the determination of kinetic parameters of recombinant CYP enzymes

 
Multiple CYP enzymes were capable of catalyzing hydroxylation of estrone. The kinetics of estrone metabolism by a suite of recombinant enzymes was determined using a substrate concentration range of 1 to 50 µmol/L and an incubation time of 5 minutes (Table 2). To allow comparison of our results with previously published results, a table of turnover at concentrations of 1, 5, and 50 µmol/L is provided (Table 3). The CYP1 family enzymes were the most active, particularly towards 2-hydroxylations and 4-hydroxylation. The apparent Km values for all detectable estrone hydroxylations by these enzymes were <10 µmol/L. CYP3A5 also catalyzed all three hydroxylations with apparent Km values of ~10 µmol/L. Addition of cytochrome b5 enhanced the activity of CYP3A5 and CYP3A4. CYP2C19 also supported the 2-hydroxylation and 16{alpha}-hydroxylation reactions.


View this table:
[in this window]
[in a new window]
  		Table 2. Apparent Km and Vmax of estrone metabolism by recombinant human CYP enzymes in vitro

 

View this table:
[in this window]
[in a new window]
  		Table 3. Metabolism of estrone by recombinant human cytochrome P450 enzymes at substrate concentrations of 1, 5, and 50 µmol/L

 
The correlation of estrone oxidation with catalytic markers of specific CYP activities was assessed using a panel of 16 adult livers, including males and females (Table 4). The formation of 2-OH-estrone correlated with the marker activities of CYP1A2 and CYP2C19 (Fig. 2, top), whereas 4-OH-estrone formation correlated only with CYP2C9-mediated marker activity and with the formation of 2-OH-estrone. The formation of 16{alpha}-OH-estrone correlated with marker activities for CYP2C19 and CYP1A2 (Fig. 2, bottom). Interpretation of these results is confounded by a correlation between CYP2C19 and CYP1A2 marker activities (Spearman's {rho} = 0.62, P = 0.01). When a substrate concentration of 2.5 µmol/L estrone was used, only 2-OH-estrone formation was identified in sufficient quantity to enable accurate correlation studies. 2-OH-estrone formation at this substrate concentration correlated with CYP1A2 ({rho} = 0.69, P = 0.02) and CYP2C9 ({rho} = 0.74, P = 0.01) marker activities. Furthermore, 2-OH-estrone formation was highly correlated with 4-OH-estrone formation but not 16{alpha}-hydroxylation. At a substrate concentration of 25 µmol/L, 2-hydroxylation and 16{alpha}-hydroxylation were correlated ({rho} = 0.75, P = 0.001).


View this table:
[in this window]
[in a new window]
  		Table 4. Spearman's correlation coefficient of estrone hydroxylations with markers of specific CYP activities

 

Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Correlation of estrone hydroxylations with marker activities in human liver microsomes. The formation of 2-OH-estrone (top) was significantly correlated with both phenacetin-O-deethylation (CYP1A2 marker activity; Spearman's {rho} = 0.83, P < 0.001) and (S)-mephenytoin-4'-hydroxylation (CYP2C19 marker activity; Spearman's {rho} = 0.60, P = 0.03). The formation of 16{alpha}-OH-estrone (bottom) was also significantly correlated with phenacetin-O-deethylation (Spearman's {rho} = 0.57, P = 0.03) and (S)-mephenytoin-4'-hydroxylation (Spearman's {rho} = 0.69, P = 0.005). Estrone hydroxylation was determined at a substrate concentration of 25 µmol/L, 1 mg/mL human liver microsomes, and a 10-minute incubation period.

 
To further explore the contribution of specific CYP forms to estrone metabolism in the liver, chemical and immunologic inhibition studies were carried out. Immunoinhibition studies were attempted with monoclonal antibodies. Unfortunately, incubations with control antibodies led to increased metabolism of low concentrations of estrone and erratic results so that no conclusive results were obtained.

Inhibition studies done at 25 µmol/L estrone substrate concentrations (Fig. 3) showed significant inhibition of 2-OH-estrone formation by the CYP1A2 inhibitor furafylline, by the CYP3A/2C9 inhibitor ketoconazole, and by the CYP2C19/CYP2C9 inhibitor omeprazole. The effect of nirvanol, a specific CYP2C19 inhibitor, was not significant, as it varied from ~35% to <3% inhibition. However, this inhibition was inversely correlated to the activity of CYP2C19 in the liver microsomes (r2 = 0.99; P < 0.01), suggesting that CYP2C19 did contribute when present in sufficient quantities. When the concentration of estrone was reduced to 2.5 µmol/L, furafylline reduced 2-OH-estrone formation to below detectable levels. Inhibition of the other two metabolites could not be accurately assessed at this low substrate concentration.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Inhibition of 2-OH-estrone (top) and 16{alpha}-OH-estrone formation by furafylline, ketoconazole, nirvanol, and omeprazole. From incubations with three to seven separate livers and expressed as percentage of the corresponding vehicle control. Inhibition was compared by one-way ANOVA and post hoc analysis with Bonferroni t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
There was significant inhibition of 16{alpha}-OH-estrone formation by the CYP2C19 inhibitors nirvanol and omeprazole. The effects of ketoconazole and furafylline were not significant Fig. 3). Ketoconazole (10 µmol/L) and furafylline (10 µmol/L) both reduced formation of 4-OH-estrone to below quantifiable concentrations at a substrate concentration of 25 µmol/L in three separate livers, whereas nirvanol had no consistent effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pharmacogenetic studies designed to assess the influence of genetic polymorphisms in metabolizing enzymes on outcomes require knowledge of the specific enzymes involved in the metabolic pathways. To gain a better understanding of the role of specific CYP enzymes in estrone metabolism, we investigated estrone metabolism at lower estrone concentrations and shorter incubations times than had previously been employed. Using this approach, we have identified a different P450 specificity profile.

The studies with recombinant enzymes showed that multiple CYP enzymes are capable of catalyzing estrone hydroxylation. Of the enzymes evaluated, CYP1A1 and CYP1B1 are not expressed in any significant quantity in the liver. Therefore, they would not be expected to contribute to the overall systemic metabolism of estrone. However, both enzymes have been identified in breast tissue (46, 47). CYP1A1 displayed relatively high activity for all three hydroxylations, suggesting that it may play an important role in extrahepatic tissues where it is expressed. Although CYP1A1 was more active with regard to 2-hydroxylation and 4-hydroxylation, it also displayed one of the greatest 16{alpha}-hydroxylating activities of the CYP enzymes tested. CYP1B1 displayed a high affinity for estrone and preferentially catalyzed 4-hydroxylation over 2-hydroxylation. CYP1B1 did not seem to contribute to 16{alpha}-hydroxylation. These results are consistent with previously published results, where CYP1B1 was shown to preferentially catalyze 4-hydroxylation, whereas CYP1A1 was shown to preferentially catalyze 2-hydroxylation (31, 48, 49). We report here higher Vmax and lower apparent Km values for 2-hydroxylation and 4-hydroxylation by both enzymes compared with previous studies (despite the same source of recombinant enzymes being used in one study; ref. 31). This is likely attributable to the shorter incubation times employed in this study, as illustrated in Table 1. Huang et al. (30) reported 16{alpha}-hydroxylation by CYP1A1 but provided no kinetic assessment, whereas no activity by CYP1B1 has been reported. It has been reported that CYP1A1 can catalyze the 16{alpha}-hydroxylation of estradiol (50).

Among the CYP enzymes expressed in the liver, CYP1A2 was the most active with regard to 2-hydroxylation. When the relative intrinsic clearances of the various enzymes were corrected for the differing expression levels in the typical human liver (51, 52), the predicted contribution from CYP1A2 was eight times higher than other CYP enzymes (Table 5). The CYP2C and CYP3A forms would be expected to have a higher contribution as the substrate concentration of estrone increased. These predictions were borne out by the correlation between 2-hydroxylation of 25 µmol/L estrone and marker activities for CYP1A2, CYP2C19, and CYP3A4 (Table 4). Inhibitors of CYP1A2, CYP3A4, and CYP2C19/2C9 were all able to inhibit the formation of 2-OH-estrone, confirming the contribution of multiple enzymes to this metabolic pathway at higher estrone concentrations. One would predict that at lower substrate concentrations, CYP1A2 would be the predominant enzyme involved. This was confirmed by the ability of furafylline to reduce 2-OH-estrone formation to below detectable levels at a substrate concentration of 2.5 µmol/L, and the loss of correlation with CYP2C19 and CYP3A4 marker activities at lower concentrations. Based on the results presented here, CYP1A2 is predominantly responsible for 2-hydroxylation of estrone, but CYP2C19, CYP3A4, and, to a lesser extent, CYP2C9 contribute as the substrate concentration increases.


View this table:
[in this window]
[in a new window]
  		Table 5. Estimated contributions of different CYP enzymes to overall hepatic clearance of estrone based on expected quantity of CYP in the liver

 
These results are consistent with previously published studies indicating an important role for CYP1A2 in the liver (31, 35, 36). Again, we observed higher velocities and lower apparent Km values than previously reported. The extent of the contribution by CYP2C19 to 2-hydroxylation of estrone has been overlooked in previous studies, either as a result of not examining the recombinant enzyme or as a result of comparing activities at high substrate concentrations.

Based on the results from the recombinant enzymes, one would predict that CYP1A2 would be the major contributor at low estrone concentrations to 4-hydroxylation of estrone, but that CYP3A4/5 (see Table 3) could contribute as estrone concentrations increased. Only the CYP2C9 marker activity correlated with 4-hydroxylation of 25 µmol/L estrone in human hepatic microsomes. However, the lack of correlation with CYP1A2 marker activities may reflect the contribution of multiple enzymes at this high substrate concentration, as furafylline and ketoconazole both inhibited the formation of 4-OH-estrone. The correlation of 4-OH-estrone formation with 2-OH-estrone at low estrone (2.5 µmol/L) concentrations is consistent with a major role for CYP1A2. Furafylline and ketoconazole both inhibited the 4-hydroxylation reaction, supporting the involvement of CYP1A2 and suggesting a contribution from CYP3A4/CYP3A5 at the substrate concentration employed. Thus, our results are consistent with a predominant role of CYP1A2 in the liver and contributions from CYP3A4/CYP3A5 at higher substrate concentrations. These results are consistent with previously published results showing a dominant role for CYP1A2 (32, 35, 36). As with 2-hydroxylation, the apparent Km was lower and the Vmax higher than in previous studies.

Of the hepatic CYP enzymes, CYP2C19 and the CYP3A5 forms were most active in the formation of 16{alpha}-OH-estrone. Using our incubation conditions, we were not able to obtain an accurate estimate of the Km and Vmax for CYP3A4, because metabolism was not detected at the lower concentrations. However, it was capable of 16{alpha}-hydroxylation at higher substrate concentrations (data not shown) or longer incubation times and showed higher activity when supplemented with cytochrome b5 (Table 3). As previously reported (32), CYP3A7 displayed 16{alpha}-hydroxylation activity (Table 3), but this enzyme is not normally expressed in significant quantities in adult tissues. From the recombinant data, CYP2C19 would be expected to be the major hepatic enzyme responsible for 16{alpha}-hydroxylation based on the relative expression of the different enzymes (Table 5). This prediction was borne out by the correlation data and the chemical inhibition studies. There was a weak correlation with CYP1A2 marker activity, but no significant inhibition by furafylline was observed; thus, the contribution of CYP1A2 was minor. In contrast, there was a significant correlation with CYP2C19 activity and a consistent inhibition by CYP2C19 inhibitors at a substrate concentration of 25 µmol/L. The weak correlation with CYP1A2 marker activity is explained by the correlation between CYP2C19 and CYP1A2. Ketoconazole showed weak, nonsignificant inhibition of 16{alpha}-OH-estrone formation, possibly indicative of a minor contribution from CYP3A4/CYP3A5 enzymes in the liver. The inhibition was not noticeably greater in livers with higher CYP3A5 expression.

Together, our results suggest that hepatic metabolism of estrone to 16{alpha}-OH-estrone is most likely mediated by CYP2C19 and extrahepatic metabolism is mediated by CYP1A1, CYP2C19, and CYP3A5, depending on the relative expression of the enzymes and the endogenous estrone concentration. These conclusions are considerably different from previously reported studies that have identified CYP3A4/CYP3A5 as the predominant enzymes responsible for 16{alpha}-hydroxylation (30, 32, 35, 36). These differences may be attributed to the differing incubation conditions employed, in particular, lower substrate concentrations and shorter incubation times. The reported apparent Km values for CYP3A4/CYP3A5 were much higher than the observed CYP3A5 Km in this study (30, 35). Previous studies have shown that CYP2C19 can catalyze 16{alpha}-hydroxylation of estrone, but kinetic studies of CYP2C19 were not done with estrone as a substrate (30, 35, 36). CYP2C9 can also catalyze the reaction (30, 35, 36), but we only observed turnover at high substrate concentrations and with prolonged incubation times (data not shown). Lee did not observe 16{alpha}-hydroxylation with either CYP2C9 or CYP2C19 (31, 32).

These results are highly significant as numerous studies have tried to link genetic polymorphisms in CYP enzymes with breast cancer risk. Our results suggest that for estrone, the critical enzymes are CYP1A1, CYP1A2, CYP1B1, CYP2C19, and CYP3A5. Genetic polymorphisms that influence expression and activity have been identified for all five of these enzymes. However, only genetic polymorphisms in CYP1A1 and CYP1B1 have been studied in association with breast cancer risk to date (53-61), with conflicting results.

Estrone and estrone sulfate are the most abundant estrogens, particularly in postmenopausal women (40), and estradiol is predominantly cleared to estrone metabolites (62). It has also been shown that estrone sulfate can act as a direct donator of estrone for CYP-dependent metabolism (30). Therefore, oxidative metabolites of estrone are the most likely candidates to influence breast cancer risk in women, particularly postmenopausal women.

From a breast cancer perspective, it is important to consider whether or not the enzymes under study are expressed in breast tissue. Several studies have addressed this issue, with somewhat conflicting results. There is general consensus that CYP1A1 and CYP1B1 are expressed in normal and malignant breast tissue, although the percentage of normal and malignant tissue expressing the enzymes varied from 25% to 100% (46, 63-66). The expression of CYP3A4, CYP3A5, and CYP2C19 are less clear. Some studies have found a significant and consistent expression of the CYP3A enzymes (63, 64, 66, 67), whereas other studies have not found any expression (65). Nonspecific probes for CYP2C forms have identified expression of CYP2C mRNA in breast tissue (65) and a CYP2C protein distinct from the common liver CYP2C protein (68). However, only one study has specifically addressed the question of CYP2C19. No expression of CYP2C19 was observed at the mRNA level; thus, the presence of this enzyme in breast tissue remains unproven (64). Similarly, the expression of CYP1A2 has not been shown in the breast (64).

These data presented here suggest that CYP1A1, CYP1B1, and CYP3A5 are likely to be the most important CYP enzymes in breast tissue. In contrast, CYP1A2 and CYP2C19 are most likely to be important in the liver, whereas their role in the breast remains unknown until further studies are done to characterize their expression in breast tissue. The probable pathways of estrone metabolism in vivo are illustrated in Fig. 4. Based on these results, the contribution of polymorphisms in CYP1A2, CYP2C19, and CYP3A5 to breast cancer risk should be assessed, in addition to the CYP1A1 and CYP1B1 polymorphisms previously studied. These studies are currently under way in our laboratory.


Figure 4
View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Postulated pathways of estrone metabolism in vivo. The probable contribution of different CYP enzymes to estrone metabolism in vivo. The question mark following CYP2C19 in breast tissue indicates that the expression of this enzyme has not been confirmed in this location.

 

   Footnotes
 
Grant support: Canadian Breast Cancer Research Initiative and the Atlantic Innovation Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A.E. Cribb holds a Canada Research Chair in comparative pharmacology and toxicology.

Received 10/17/05; revised 12/22/05; accepted 1/16/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Liehr JG. Dual role of oestrogens as hormones and pro-carcinogens: tumour initiation by metabolic activation of oestrogens. Eur J Cancer Prev 1997;6:3–10.[CrossRef][Medline]
  2. Zumoff B. Does postmenopausal estrogen administration increase the risk of breast cancer? Contributions of animal, biochemical, and clinical investigative studies to a resolution of the controversy. Proc Soc Exp Biol Med 1998;217:30–7.[CrossRef][Medline]
  3. Veronesi U, Maisonneuve P, Costa A, et al. Prevention of breast cancer with tamoxifen: preliminary findings from the Italian randomised trial among hysterectomised women. Italian Tamoxifen Prevention Study. Lancet 1998;352:93–7.[Medline]
  4. Russo J, Hasan Lareef M, Balogh G, Guo S, Russo IH. Estrogen and its metabolites are carcinogenic agents in human breast epithelial cells. J Steroid Biochem Mol Biol 2003;87:1–25.[CrossRef][Medline]
  5. Yager JD, Liehr JG. Molecular mechanisms of estrogen carcinogenesis. Annu Rev Pharmacol Toxicol 1996;36:203–32.[CrossRef][Medline]
  6. Liehr JG, Fang WF, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian hamsters. J Steroid Biochem 1986;24:353–6.[CrossRef][Medline]
  7. Cavalieri EL, Rogan EG. A unifying mechanism in the initiation of cancer and other diseases by catechol quinones. Ann N Y Acad Sci 2004;1028:247–57.[CrossRef][Medline]
  8. Bolton JL, Pisha E, Zhang F, Qiu S. Role of quinoids in estrogen carcinogenesis. Chem Res Toxicol 1998;11:1113–27.[CrossRef][Medline]
  9. Liehr JG, Ulubelen AA, Strobel HW. Cytochrome P-450-mediated redox cycling of estrogens. J Biol Chem 1986;261:16865–70.[Abstract/Free Full Text]
  10. Liehr JG. Genotoxic effects of estrogens. Mutat Res 1990;238:269–76.[Medline]
  11. Liehr JG, Roy D. Free radical generation by redox cycling of estrogens. Free Radic Biol Med 1990;8:415–23.[CrossRef][Medline]
  12. Bradlow HL, Telang NT, Sepkovic DW, Osborne MP. 2-Hydroxyestrone: the ‘good’ estrogen. J Endocrinol 1996;150 Suppl:S259–65.[Medline]
  13. Barnea ER, MacLusky NJ, Naftolin F. Kinetics of catechol estrogen-estrogen receptor dissociation: a possible factor underlying differences in catechol estrogen biological activity. Steroids 1983;41:643–56.[CrossRef][Medline]
  14. Van Aswegen CH, Purdy RH, Wittliff JL. Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers. J Steroid Biochem 1989;32:485–92.[CrossRef][Medline]
  15. Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 1998;19:1–27.[Abstract/Free Full Text]
  16. Lottering ML, Haag M, Seegers JC. Effects of 17 ß-estradiol metabolites on cell cycle events in MCF-7 cells. Cancer Res 1992;52:5926–32.[Abstract/Free Full Text]
  17. Schutze N, Vollmer G, Tiemann I, Geiger M, Knuppen R. Catecholestrogens are MCF-7 cell estrogen receptor agonists. J Steroid Biochem Mol Biol 1993;46:781–9.[CrossRef][Medline]
  18. Vandewalle B, Lefebvre J. Opposite effects of estrogen and catecholestrogen on hormone-sensitive breast cancer cell growth and differentiation. Mol Cell Endocrinol 1989;61:239–46.[CrossRef][Medline]
  19. Bradlow HL, Hershcopf RJ, Martucci CP, Fishman J. Estradiol 16 {alpha}-hydroxylation in the mouse correlates with mammary tumor incidence and presence of murine mammary tumor virus: a possible model for the hormonal etiology of breast cancer in humans. Proc Natl Acad Sci U S A 1985;82:6295–9.[Abstract/Free Full Text]
  20. Kabat GC, Chang CJ, Sparano JA, et al. Urinary estrogen metabolites and breast cancer: a case-control study. Cancer Epidemiol Biomarkers Prev 1997;6:505–9.[Abstract]
  21. Telang NT, Suto A, Bradlow HL, Wong GY, Osborne MP. Genotoxic damage and aberrant proliferation in mouse mammary epithelial cells. Recent Prog Horm Res 1993;48:481–8.[Medline]
  22. Telang NT, Suto A, Wong GY, Osborne MP, Bradlow HL. Induction by estrogen metabolite 16 {alpha}-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells. J Natl Cancer Inst 1992;84:634–8.[Abstract/Free Full Text]
  23. Swaneck GE, Fishman J. Covalent binding of the endogenous estrogen 16 {alpha}-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization. Proc Natl Acad Sci U S A 1988;85:7831–5.[Abstract/Free Full Text]
  24. Fowke JH, Qi D, Bradlow HL, et al. Urinary estrogen metabolites and breast cancer: differential pattern of risk found with pre- versus post-treatment collection. Steroids 2003;68:65–72.[CrossRef][Medline]
  25. Adlercreutz H, Gorbach SL, Goldin BR, et al. Estrogen metabolism and excretion in Oriental and Caucasian women. J Natl Cancer Inst 1994;86:1076–82.[Abstract/Free Full Text]
  26. Meilahn EN, De Stavola B, Allen DS, et al. Do urinary oestrogen metabolites predict breast cancer? Guernsey III cohort follow-up. Br J Cancer 1998;78:1250–5.[Medline]
  27. Schneider J, Kinne D, Fracchia A, et al. Abnormal oxidative metabolism of estradiol in women with breast cancer. Proc Natl Acad Sci U S A 1982;79:3047–51.[Abstract/Free Full Text]
  28. Lee AJ, Zhu BT. NADPH-dependent formation of polar and nonpolar estrogen metabolites following incubations of 17 ß-estradiol with human liver microsomes. Drug Metab Dispos 2004;32:876–83.[Abstract/Free Full Text]
  29. Hayes CL, Spink DC, Spink BC, et al. 17 ß-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc Natl Acad Sci U S A 1996;93:9776–81.[Abstract/Free Full Text]
  30. Huang Z, Guengerich FP, Kaminsky LS. 16{alpha}-Hydroxylation of estrone by human cytochrome P4503A4/5. Carcinogenesis 1998;19:867–72.[Abstract/Free Full Text]
  31. Lee AJ, Cai MX, Thomas PE, Conney AH, Zhu BT. Characterization of the oxidative metabolites of 17ß-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. Endocrinology 2003;144:3382–98.[CrossRef][Medline]
  32. Lee AJ, Conney AH, Zhu BT. Human cytochrome P450 3A7 has a distinct high catalytic activity for the 16{alpha}-hydroxylation of estrone but not 17ß-estradiol. Cancer Res 2003;63:6532–6.[Abstract/Free Full Text]
  33. Lee AJ, Kosh JW, Conney AH, Zhu BT. Characterization of the NADPH-dependent metabolism of 17ß-estradiol to multiple metabolites by human liver microsomes and selectively expressed human cytochrome P450 3A4 and 3A5. J Pharmacol Exp Ther 2001;298:420–32.[Abstract/Free Full Text]
  34. Martucci CP, Fishman J. P450 enzymes of estrogen metabolism. Pharmacol Ther 1993;57:237–57.[CrossRef][Medline]
  35. Yamazaki H, Shaw PM, Guengerich FP, Shimada T. Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes. Chem Res Toxicol 1998;11:659–65.[CrossRef][Medline]
  36. Shou M, Korzekwa KR, Brooks EN, et al. Role of human hepatic cytochrome P450 1A2 and 3A4 in the metabolic activation of estrone. Carcinogenesis 1997;18:207–14.[Abstract/Free Full Text]
  37. Zhu BT, Lee AJ. NADPH-dependent metabolism of 17ß-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms. Steroids 2005;70:225–44.[CrossRef][Medline]
  38. van Landeghem AA, Poortman J, Nabuurs M, Thijssen JH. Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue. Cancer Res 1985;45:2900–6.[Abstract/Free Full Text]
  39. Castagnetta L, Granata OM, Cocciadiferro L, et al. Sex steroids, carcinogenesis, and cancer progression. Ann N Y Acad Sci 2004;1028:233–46.[CrossRef][Medline]
  40. Gruber CJ, Tschugguel W, Schneeberger C, Huber JC. Production and actions of estrogens. N Engl J Med 2002;346:340–52.[Free Full Text]
  41. Chatterton RT, Jr., Geiger AS, Mateo ET, Helenowski IB, Gann PH. Comparison of hormone levels in nipple aspirate fluid of pre- and postmenopausal women: effect of oral contraceptives and hormone replacement. J Clin Endocrinol Metab 2005;90:1686–91.[Abstract/Free Full Text]
  42. Chatterton RT, Jr., Geiger AS, Khan SA, et al. Variation in estradiol, estradiol precursors, and estrogen-related products in nipple aspirate fluid from normal premenopausal women. Cancer Epidemiol Biomarkers Prev 2004;13:928–35.[Abstract/Free Full Text]
  43. Busby WF, Jr., Ackermann JM, Crespi CL. Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 1999;27:246–9.[Abstract/Free Full Text]
  44. Chauret N, Gauthier A, Nicoll-Griffith DA. Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab Dispos 1998;26:1–4.[Abstract/Free Full Text]
  45. Hickman D, Wang JP, Wang Y, Unadkat JD. Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos 1998;26:207–15.[Abstract/Free Full Text]
  46. McFadyen MC, Breeman S, Payne S, et al. Immunohistochemical localization of cytochrome P450 CYP1B1 in breast cancer with monoclonal antibodies specific for CYP1B1. J Histochem Cytochem 1999;47:1457–64.[Abstract/Free Full Text]
  47. Moutsatsou P, Fountas L, Coulocheri S, Kassi E, Sekeris CE. The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells. J Cancer Res Clin Oncol 1999;125:214–8.[Medline]
  48. Shimada T, Watanabe J, Kawajiri K, et al. Catalytic properties of polymorphic human cytochrome P450 1B1 variants. Carcinogenesis 1999;20:1607–13.[Abstract/Free Full Text]
  49. Kisselev P, Schunck WH, Roots I, Schwarz D. Association of CYP1A1 polymorphisms with differential metabolic activation of 17ß-estradiol and estrone. Cancer Res 2005;65:2972–8.[Abstract/Free Full Text]
  50. Badawi AF, Cavalieri EL, Rogan EG. Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16{alpha}-hydroxylation of 17ß-estradiol. Metabolism 2001;50:1001–3.[CrossRef][Medline]
  51. Rodrigues AD. Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 1999;57:465–80.[CrossRef][Medline]
  52. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 1994;270:414–23.[Abstract/Free Full Text]
  53. Bailey LR, Roodi N, Verrier CS, et al. Breast cancer and CYPIA1, GSTM1, and GSTT1 polymorphisms: evidence of a lack of association in Caucasians and African Americans. Cancer Res 1998;58:65–70.[Abstract/Free Full Text]
  54. Dialyna IA, Arvanitis DA, Spandidos DA. Genetic polymorphisms and transcriptional pattern analysis of CYP1A1, AhR, GSTM1, GSTP1 and GSTT1 genes in breast cancer. Int J Mol Med 2001;8:79–87.[Medline]
  55. Mitrunen K, Hirvonen A. Molecular epidemiology of sporadic breast cancer. The role of polymorphic genes involved in oestrogen biosynthesis and metabolism. Mutat Res 2003;544:9–41.[CrossRef][Medline]
  56. Taioli E, Bradlow HL, Garbers SV, et al. Role of estradiol metabolism and CYP1A1 polymorphisms in breast cancer risk. Cancer Detect Prev 1999;23:232–7.[CrossRef][Medline]
  57. Taioli E, Trachman J, Chen X, Toniolo P, Garte SJ. A CYP1A1 restriction fragment length polymorphism is associated with breast cancer in African-American women. Cancer Res 1995;55:3757–8.[Abstract/Free Full Text]
  58. De Vivo I, Hankinson SE, Li L, Colditz GA, Hunter DJ. Association of CYP1B1 polymorphisms and breast cancer risk. Cancer Epidemiol Biomarkers Prev 2002;11:489–92.[Abstract/Free Full Text]
  59. Dunning AM, Dowsett M, Healey CS, et al. Polymorphisms associated with circulating sex hormone levels in postmenopausal women. J Natl Cancer Inst 2004;96:936–45.[Abstract/Free Full Text]
  60. Rylander-Rudqvist T, Wedren S, Granath F, et al. Cytochrome P450 1B1 gene polymorphisms and postmenopausal breast cancer risk. Carcinogenesis 2003;24:1533–9.[Abstract/Free Full Text]
  61. Saintot M, Malaveille C, Hautefeuille A, Gerber M. Interactions between genetic polymorphism of cytochrome P450–1B1, sulfotransferase 1A1, catechol-o-methyltransferase and tobacco exposure in breast cancer risk. Int J Cancer 2003;107:652–7.[CrossRef][Medline]
  62. Fishman J. Aromatic hydroxylation of estrogens. Annu Rev Physiol 1983;45:61–72.[CrossRef][Medline]
  63. El-Rayes BF, Ali S, Heilbrun LK, et al. Cytochrome p450 and glutathione transferase expression in human breast cancer. Clin Cancer Res 2003;9:1705–9.[Abstract/Free Full Text]
  64. Modugno F, Knoll C, Kanbour-Shakir A, Romkes M. A potential role for the estrogen-metabolizing cytochrome P450 enzymes in human breast carcinogenesis. Breast Cancer Res Treat 2003;82:191–7.[CrossRef][Medline]
  65. Iscan M, Klaavuniemi T, Coban T, et al. The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue. Breast Cancer Res Treat 2001;70:47–54.[CrossRef][Medline]
  66. Schmidt R, Baumann F, Knupfer H, et al. CYP3A4, CYP2C9 and CYP2B6 expression and ifosfamide turnover in breast cancer tissue microsomes. Br J Cancer 2004;90:911–6.[CrossRef][Medline]
  67. Kapucuoglu N, Coban T, Raunio H, et al. Expression of CYP3A4 in human breast tumour and non-tumour tissues. Cancer Lett 2003;202:17–23.[CrossRef][Medline]
  68. Forrester LM, Hayes JD, Millis R, et al. Expression of glutathione S-transferases and cytochrome P450 in normal and tumor breast tissue. Carcinogenesis 1990;11:2163–70.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
M. N. Timofeeva, S. Kropp, W. Sauter, L. Beckmann, A. Rosenberger, T. Illig, B. Jager, K. Mittelstrass, H. Dienemann, The LUCY-Consortium, et al.
CYP450 polymorphisms as risk factors for early-onset lung cancer: gender-specific differences
Carcinogenesis, July 1, 2009; 30(7): 1161 - 1169.
[Abstract] [Full Text] [PDF]

Home page
 BioinformaticsHome page
J. Hu
Cancer outlier detection based on likelihood ratio test
Bioinformatics, October 1, 2008; 24(19): 2193 - 2199.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. Canonico, E. Bouaziz, L. Carcaillon, C. Verstuyft, A. Guiochon-Mantel, L. Becquemont, P.-Y. Scarabin, and for the Estrogen Thromboembolism Risk (ESTHER) Stu
Synergism between Oral Estrogen Therapy and Cytochrome P450 3A5*1 Allele on the Risk of Venous Thromboembolism among Postmenopausal Women
J. Clin. Endocrinol. Metab., August 1, 2008; 93(8): 3082 - 3087.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
T. R. Rebbeck, A. B. Troxel, A. H. Walker, S. Panossian, S. Gallagher, E. G. Shatalova, R. Blanchard, S. Norman, G. Bunin, A. DeMichele, et al.
Pairwise Combinations of Estrogen Metabolism Genotypes in Postmenopausal Breast Cancer Etiology
Cancer Epidemiol. Biomarkers Prev., March 1, 2007; 16(3): 444 - 450.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cribb, A. E.
Right arrow Articles by Saleh, T. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cribb, A. E.
Right arrow Articles by Saleh, T. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online