Hitomi Takemura

Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro.

In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).

Selective inhibition of methoxyflavonoids on human CYP1B1 activity

Cytochrome P450 (CYP) 1B1 catalyzes 17beta-estradiol (E(2)) to predominantly carcinogenic 4-hydroxy-E(2), whereas CYP1A1 and 1A2 convert E(2) to non-carcinogenic 2-hydroxy-E(2). Hence, selective inhibition of CYP1B1 is recognized to be beneficial for the prevention of E(2) related breast cancer. In this study, we first evaluated the structure-property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin O-deethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between C-positions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited CYP1B1 activity. In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1. To understand why selective inhibition was observed, we carried out a molecular docking analysis of these methoxyflavonoids with the 2-3 double bond and CYP1s. The results suggested that chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser-122 in CYP1A1 and Thr-124 in CYP1A2. In conclusion, our results demonstrate: (1) strong inhibitory effects of flavonoids on CYP1 activities require the 2-3 double bond on the C-ring; (2) methoxyflavonoids with the 2-3 double bond had strong and selective inhibition against CYP1B1, suggesting chemopreventive flavonoids for E(2) related breast cancer; and (3) binding specificity of these methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues.

Breast cancer and flavonoids - a role in prevention.

Endogenous estrogens, such as 17β-estradiol (E2), are implicated in the development of breast cancer. The putative mechanisms by which estrogens exert the carcinogenic effects have been recognized to involve the redox cycling of estrogen metabolites and subsequent estrogen-DNA adduct formation as well as the estrogen receptor-dependent pathway of estrogen-induced cell growth. The former pathway is regulated by phase I enzymes, mainly cytochrome P450 (CYP) 1A1, 1A2, and 1B1. Among them, CYP1B1 predominantly catalyzes the C4-position of E2 and forms carcinogenic 4-hydroxy-E2 (4-OHE2), whereas CYP1A1 and CYP1A2 convert E2 to noncarcinogenic 2-hydroxy-E2. Formed 4-OHE2 is further oxidized to semiquinones and quinones, which form DNA adducts, leading to mutagenic lesions. Consequently, CYP1B1 is highly expressed, and 4-OHE2 is predominantly detected in estrogen target neoplastic tissues. Moreover, invasion and metastasis are also involved in the development of breast cancer. Epidemiological studies suggest an inverse association between a higher intake of flavonoids and breast cancer risk. Flavonoids, which are widely distributed in the plant kingdom, have been recently reported as candidate compounds that can exert chemopreventive effects in estrogen-dependent or independent breast cancer. In this review, we provide a comprehensive overview of breast cancer and chemoprevention by flavonoids, mainly focusing on ER-mediated hormonal regulation, redox cycling of estrogen metabolites, and selective inhibition of CYP1B1.

A 3D model of CYP1B1 explains the dominant 4-hydroxylation of estradiol.

CYP1A1 and CYP1A2 exhibit catalytic activity predominantly for the 2-hydroxylation of estradiol, whereas CYP1B1 exhibits catalytic activity predominantly for 4-hydroxylation of estradiol. To understand why CYP1B1 predominantly hydroxylates the 4-position of estradiol, we constructed three-dimensional structures of CYP1A1 and CYP1B1 by homology modeling, using the crystal structure of CYP1A2, and studied the docking mode of estradiol with CYP1A1, CYP1A2, and CYP1B1. The results demonstrated that two particular amino acid residues for each CYP, namely Thr124 and Phe260 of CYP1A2, Ser122 and Phe258 of CYP1A1, and Ala133 and Asn265 of CYP1B1, play an important role in estradiol recognition.

Inhibitory effects of chrysoeriol on DNA adduct formation with benzo[a]pyrene in MCF-7 breast cancer cells

Cytochrome P450 (CYP) 1 families including CYP1A1, 1A2 and 1B1 are well known to be deeply involved in the initiation of several cancers, due to the fact that they activate environmental pro-carcinogens to form ultimate carcinogens. Benzo[a]pyrene (BaP) is one of the major classes of prototypical pro-carcinogen. It is activated by the CYP1 family to its ultimate carcinogenic forms, mainly BaP-7,8-diol-9,10-epoxide (BPDE), and it forms adducts with DNA. This has been recognized to be a major initiation pathway for cancer. Our previous study demonstrated that chrysoeriol, which is a dietary methoxyflavonoid, selectively inhibited CYP1B1 enzymatic activity and might protect the CYP1B1 related-diseases such as breast cancer. In the present study, we further examined the effects of chrysoeriol on the other initiation pathway of cancer relating to the CYP1 family with BaP in human breast cancer MCF-7 cells. The effects of chrysoeriol on the formation of BPDE-DNA adducts were analyzed specifically using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. When MCF-7 cells were incubated with 2 microM BaP for 24h, three types of BPDE-dG adducts, especially (+)-trans-BPDE-dG as the dominant adduct, were detected. Co-treatment of MCF-7 cells with 10 microM chrysoeriol and BaP remarkably reduced (+)-trans-BPDE-dG formation. Chrysoeriol (1-10 microM) dose-dependently inhibited both EROD activity and the gene expressions of CYP1A1, 1B1 and 1A2 stimulated by treatment with BaP. In addition, the same amounts of chrysoeriol significantly inhibited the binding of BaP to the aryl hydrocarbon receptor (AhR), which is the key factor concerning the induction of the CYP1 families. In conclusion, our results clearly indicate that chrysoeriol inhibited the formation of BPDE-DNA adducts via regulation of the AhR pathway stimulated by BaP. As a consequence chrysoeriol may be involved in the chemoprevention of environmental pro-carcinogens such as BaP.

Quercetin-3-O-glucronide inhibits noradrenaline binding to α2-adrenergic receptor, thus suppressing DNA damage induced by treatment with 4-hydroxyestradiol and noradrenaline in MCF-10A cells

Risk factors for breast cancer include estrogens such as 17β-estradiol (E2) and high stress levels. 4-Hydroxyestradiol (4-OHE2), a metabolite of E2 formed preferentially by cytochrome P450 1B1, is oxidized to E2-3,4-quinone, which reacts with DNA to form depurinating adducts that exert genotoxicity and carcinogenicity. Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. Here, we found that treatment with 4-OHE2 (3 μM) and NA (3 nM) significantly induced the phosphorylation of histone H2AX (γ-H2AX), one of the earliest indicators of DNA damage, and apurinic (AP) sites via the α2-adrenergic receptor (α2-AR) in human mammary epithelial MCF-10A cells. As an inverse association between a higher intake of flavonoids and breast cancer risk has previously been suggested from epidemiological studies, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin in the blood, on 4-OHE2- and NA-induced γ-H2AX and AP sites. Q3G (0.1 μM) suppressed their induction and inhibited the binding of [(3)H]-NA to α2-AR. These results suggest that Q3G acts as an α2-AR antagonist and that it could be used as a chemopreventive agent for stress-promoted breast cancer.

Inhibitory Effect of Tea Extracts on the Formation of Heterocyclic Amines During Cooking of Hamburger

Tea extracts from green tea, black tea, polei tea, and rooibos tea showed an inhibitory effect on the formation of mutagenic and carcinogenic heterocyclic amines, such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-arnino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), during cooking of hamburger. In the case of green tea samples, the catechin fraction and powder reduced the MelQx content to 54% and 26% of the control, respectively. PhIP content was also suppressed to 7.0%-19.8% of the control by the addition of crude extract or a catechin fraction or powder. Catechins con-taining (-)epigallocatechin gallate and flavonoids were useful components in these tea samples, and they may act mainly as antioxidants and radical-scavenging agents.