Estrogen metabolism and exposure in a genotypic-phenotypic model for breast cancer risk prediction.

BACKGROUND: Current models of breast cancer risk prediction do not directly reflect mammary estrogen metabolism or genetic variability in exposure to carcinogenic estrogen metabolites. METHODS: We developed a model that simulates the kinetic effect of genetic variants of the enzymes CYP1A1, CYP1B1, and COMT on the production of the main carcinogenic estrogen metabolite, 4-hydroxyestradiol (4-OHE(2)), expressed as area under the curve metric (4-OHE(2)-AUC). The model also incorporates phenotypic factors (age, body mass index, hormone replacement therapy, oral contraceptives, and family history), which plausibly influence estrogen metabolism and the production of 4-OHE(2). We applied the model to two independent, population-based breast cancer case-control groups, the German GENICA study (967 cases, 971 controls) and the Nashville Breast Cohort (NBC; 465 cases, 885 controls). RESULTS: In the GENICA study, premenopausal women at the 90th percentile of 4-OHE(2)-AUC among control subjects had a risk of breast cancer that was 2.30 times that of women at the 10th control 4-OHE(2)-AUC percentile (95% CI: 1.7-3.2, P = 2.9 × 10(-7)). This relative risk was 1.89 (95% CI: 1.5-2.4, P = 2.2 × 10(-8)) in postmenopausal women. In the NBC, this relative risk in postmenopausal women was 1.81 (95% CI: 1.3-2.6, P = 7.6 × 10(-4)), which increased to 1.83 (95% CI: 1.4-2.3, P = 9.5 × 10(-7)) when a history of proliferative breast disease was included in the model. CONCLUSIONS: The model combines genotypic and phenotypic factors involved in carcinogenic estrogen metabolite production and cumulative estrogen exposure to predict breast cancer risk. IMPACT: The estrogen carcinogenesis-based model has the potential to provide personalized risk estimates.

Estrogen metabolism and breast cancer: a risk model.

Oxidative metabolites of estrogens have been implicated in the development of breast cancer, yet relatively little is known about the metabolism of estrogens in the normal breast. We developed an experimental in vitro model of mammary estrogen metabolism in which we combined purified, recombinant phase I enzymes CYP1A1 and CYP1B1 with the phase II enzymes COMT and GSTP1 to determine how 17beta-estradiol (E(2)) is metabolized. We employed both gas and liquid chromatography with mass spectrometry to measure the parent hormone E(2) as well as eight metabolites, that is, the catechol estrogens, methoxyestrogens, and estrogen-GSH conjugates. We used these experimental data to develop an in silico model, which allowed the kinetic simulation of converting E(2) into eight metabolites. The simulations showed excellent agreement with experimental results and provided a quantitative assessment of the metabolic interactions. Using rate constants of genetic variants of CYP1A1, CYP1B1, and COMT, the model further allowed examination of the kinetic impact of enzyme polymorphisms on the entire metabolic pathway, including the identification of those haplotypes producing the largest amounts of catechols and quinones. Application of the model to a breast cancer case-control population defined the estrogen quinone E(2)-3,4-Q as a potential risk factor and identified a subset of women with an increased risk of breast cancer based on their enzyme haplotypes and consequent E(2)-3,4-Q production. Our in silico model integrates diverse types of data and offers the exciting opportunity for researchers to combine metabolic and genetic data in assessing estrogenic exposure in relation to breast cancer risk.

Carcinogen-induced histone alteration in normal human mammary epithelial cells.

Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation.

The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17beta-estradiol (E2) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E2 in the presence of 2'-deoxyguanosine or 2'-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E2, the 2- and 4-catechol estrogens (2-OHE2, 4-OHE2), and the depurinating adducts 4-OHE(2)-1(alpha,beta)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE(2)-1(alpha,beta)-N3-adenine (4-OHE2-N3-Ade). CYP1B1 oxidized E2 to the catechol 4-OHE2 and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE2-N7-Gua adduct (Km, 4.6+/-0.7 micromol/L; kcat, 45+/-1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (Km, 4.6+/-1.0 micromol/L; kcat, 30+/-1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE2 production. The results present direct proof of CYP1B1-mediated, E2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis.

Estrogens, enzyme variants, and breast cancer: a risk model.

Oxidative metabolites of estrogens have been implicated in the development of breast cancer, yet relatively little is known about the metabolism of estrogens in the normal breast. We developed a mathematical model of mammary estrogen metabolism based on the conversion of 17beta-estradiol (E(2)) by the enzymes cytochrome P450 (CYP) 1A1 and CYP1B1, catechol-O-methyltransferase (COMT), and glutathione S-transferase P1 into eight metabolites [i.e., two catechol estrogens, 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)); three methoxyestrogens, 2-methoxyestradiol, 2-hydroxy-3-methoxyestradiol, and 4-methoxyestradiol; and three glutathione (SG)-estrogen conjugates, 2-OHE(2)-1-SG, 2-OHE(2)-4-SG, and 4-OHE(2)-2-SG]. When used with experimentally determined rate constants with purified enzymes, the model provides for a kinetic analysis of the entire metabolic pathway. The predicted concentration of each metabolite during a 30-minute reaction agreed well with the experimentally derived results. The model also enables simulation for the transient quinones, E(2)-2,3-quinone (E(2)-2,3-Q) and E(2)-3,4-quinone (E(2)-3,4-Q), which are not amenable to direct quantitation. Using experimentally derived rate constants for genetic variants of CYP1A1, CYP1B1, and COMT, we used the model to simulate the kinetic effect of enzyme polymorphisms on the pathway and identified those haplotypes generating the largest amounts of catechols and quinones. Application of the model to a breast cancer case-control population identified a subset of women with an increased risk of breast cancer based on their enzyme haplotypes and consequent E(2)-3,4-Q production. This in silico model integrates both kinetic and genomic data to yield a comprehensive view of estrogen metabolomics in the breast. The model offers the opportunity to combine metabolic, genetic, and lifetime exposure data in assessing estrogens as a breast cancer risk factor.

In vitro model of mammary estrogen metabolism: structural and kinetic differences between catechol estrogens 2- and 4-hydroxyestradiol.

Estrogens and their oxidative metabolites, the catechol estrogens, have been implicated in the development of breast cancer; yet, relatively little is known about estrogen metabolism in the breast. To determine how the parent hormone, 17 beta-estradiol (E(2)), is metabolized, we used recombinant, purified phase I enzymes, cytochrome P450 (CYP) 1A1 and 1B1, with the phase II enzymes catechol-O-methyltransferase (COMT) and glutathione S-transferase P1 (GSTP1), all of which are expressed in breast tissue. We employed both gas and liquid chromatography with mass spectrometry to measure E(2), the catechol estrogens 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), as well as methoxyestrogens and estrogen-GSH conjugates. The oxidation of E(2) to 2-OHE(2) and 4-OHE(2) was exclusively regulated by CYP1A1 and 1B1, regardless of the presence or concentration of COMT and GSTP1. COMT generated two products, 2-methoxyestradiol and 2-hydroxy-3-methoxyestradiol, from 2-OHE(2) but only one product, 4-methoxyestradiol, from 4-OHE(2). Similarly, GSTP1 yielded two conjugates, 2-OHE(2)-1-SG and 2-OHE(2)-4-SG, from the corresponding quinone 2-hydroxyestradiol-quinone and one conjugate, 4-OHE(2)-2-SG, from 4-hydroxyestradiol-quinone. Using the experimental data, we developed a multicompartment kinetic model for the oxidative metabolism of the parent hormone E(2), which revealed significant differences in rate constants for its C-2 and C-4 metabolites. The results demonstrated a tightly regulated interaction of phase I and phase II enzymes, in which the latter decreased the concentration of catechol estrogens and estrogen quinones, thereby reducing the potential of these oxidative estrogen metabolites to induce DNA damage.

Association of homozygous wild-type glutathione S-transferase M1 genotype with increased breast cancer risk.

More than 500 studies have examined the association of the glutathione S-transferase M1 (GSTM1) genotype with various malignancies yielding inconsistent results. The genotyping was based on a PCR assay that identified the GSTM1 null (-/-) genotype but did not distinguish homozygous wild-type (+/+) and heterozygous (+/-) individuals. We developed an assay that allowed the definition of +/+, +/-, and -/- genotypes by separate identification of wild-type and null alleles, which were found with frequencies of 0.225 and 0.775, respectively, in Caucasian women. We applied the new assay to a breast cancer case-control study and identified the +/+ genotype in 14 (6.9%) of 202 control subjects compared with 37 (18.2%) of 203 patients. Compared with women with the -/- genotype, the relative risk of breast cancer for the +/+ genotype was 2.83 (95% confidence interval, 1.45-5.59; P = 0.002), suggesting a protective effect of the GSTM1 deletion.

Sequential action of phase I and II enzymes cytochrome p450 1B1 and glutathione S-transferase P1 in mammary estrogen metabolism.

The Phase I enzyme cytochrome p450 1B1 (CYP1B1) has been postulated to play a key role in estrogen-induced mammary carcinogenesis by catalyzing the oxidative metabolism of 17beta-estradiol (E(2)) to catechol estrogens (2-OHE(2) and 4-OHE(2)) and highly reactive estrogen quinones (E(2)-2,3-Q and E(2)-3,4-Q). The potential of the quinones to induce mutagenic DNA lesions is expected to be decreased by their conjugation with glutathione (GSH) either nonenzymatically or catalyzed by glutathione S-transferase P1 (GSTP1), a Phase II enzyme. Because the interaction of the Phase I and Phase II enzymes is not well defined in this setting, we prepared recombinant purified CYP1B1 and GSTP1 to examine their individual and combined roles in the oxidative pathway and used gas and liquid chromatography/mass spectrometry to measure the parent hormone E(2), the catechol estrogens, and the GSH conjugates. 2-OHE(2) and 4-OHE(2) did not form conjugates with GSH alone or in the presence of GSTP1. However, incubation of GSH and CYP1B1 with 2-OHE(2) resulted in nearly linear conjugation through C-4 and C-1 (i.e., 2-OHE(2)-4-SG and 2-OHE(2)-1-SG), whereas the reaction of 4-OHE(2) yielded only 4-OHE(2)-2-SG. When CYP1B1 and GSTP1 were added together, the rate of conjugation was accelerated with a hyperbolic pattern of product formation in the order 4-OHE(2)-2-SG > 2-OHE(2)-4-SG >> 2-OHE(2)-1-SG. Incubation of E(2) with CYP1B1 and GSTP1 resulted in the formation of 4-OHE(2), 2-OHE(2), 4-OHE(2)-2-SG, 2-OHE(2)-4-SG, and 2-OHE(2)-1-SG. The production of GSH-estrogen conjugates was dependent on the concentrations of E(2) and GSTP1 but overall yielded only one-tenth of the catechol estrogen production. The concentration gap between catechol estrogens and GSH-estrogen conjugates may result from nonenzymatic reaction of the labile quinones with other nucleophiles besides GSH or may reflect the lower efficiency of GSTP1 compared with CYP1B1. In summary, both reactions are coordinated qualitatively in terms of product formation and substrate utilization, but the quantitative gap would leave room for the accumulation of estrogen quinones and their potential for DNA damage as part of estrogen-induced mammary carcinogenesis.

Methoxyestrogens exert feedback inhibition on cytochrome P450 1A1 and 1B1.

Cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) catalyze the oxidative metabolism of 17 beta-estradiol (E2) to catechol estrogens (2-OHE2 and 4-OHE2) and estrogen quinones, which may lead to DNA damage. Catechol-O-methyltransferase catalyzes the methylation of catechol estrogens to methoxyestrogens (2-MeOE2, 2-OH-3-MeOE2, and 4-MeOE2), which simultaneously lowers the potential for DNA damage and increases the concentration of 2-MeOE2, an antiproliferative metabolite. In this study, we showed that CYP1A1 and CYP1B1 recognized as substrates both the parent hormone E2 and the methoxyestrogens. Using purified recombinant enzymes, we demonstrated that CYP1A1 and CYP1B1 O-demethylated the methoxyestrogens to catechol estrogens according to Michaelis-Menten kinetics. Both CYP1A1 and CYP1B1 demethylated 2-MeOE2 and 2-OH-3-MeOE2 to 2-OHE2, whereas CYP1B1 additionally demethylated 4-MeOE2 to 4-OHE2. Because the P450-mediated oxidation of E2 and the O-demethylation of methoxyestrogens both yielded identical catechol estrogens as products, we used deuterated E2 (E2-d4), unlabeled methoxyestrogens, and gas chromatography/mass spectrometry to examine both reactions simultaneously. Kinetic analysis revealed that methoxyestrogens acted as noncompetitive inhibitors of E2 oxidation with K(i) ranging from 27 to 153 micro M. For both enzymes, the order of inhibition by methoxyestrogens was 2-OH-3-MeOE2 > or = 2-MeOE2 > 4-MeOE2. Thus, methoxyestrogens exert feedback inhibition on CYP1A1- and CYP1B1-mediated oxidative estrogen metabolism, thereby reducing the potential for estrogen-induced DNA damage.