- Nitidine chloride-induced CYP1 enzyme inhibition and alteration of estradiol metabolism
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The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 5 0.28±0.06 and 0.32±0.02 mM, respectively) in a concentration- dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 mM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a Ki value of 0.47±0.05 mM, whereas NC caused a mixed type of inhibition on CYP1A1 with Ki and KI values of 0.14±0.04 and 0.19±0.09 mM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NCwas a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4- OHE2-induced genotoxicity.
- Mao, Xu,Wang, Jian,Wang, Qian,Yang, Lan,Li, Yilin,Lin, Hao,Peng, Ying,Zheng, Jiang
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p. 919 - 927
(2019/09/03)
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- Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids
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We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Δ2-4)CYP1B1 and (Δ2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Δ2-4)CYP1B1 than the (Δ2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.
- Jang, Hyun-Hee,Kim, Dong-Hyun,Ahn, Taeho,Yun, Chul-Ho
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experimental part
p. 143 - 150
(2010/12/19)
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- A methoxyflavonoid, chrysoeriol, selectively inhibits the formation of a carcinogenic estrogen metabolite in MCF-7 breast cancer cells
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A 17β-estradiol (E2) is hydrolyzed to 2-hydroxy-E2 (2-OHE2) and 4-hydroxy-E2 (4-OHE2) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE2 is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3′-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E2 hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE2, which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E2 without any effects on the formation of 2-methoxy E2. In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE2 from E2..
- Takemura, Hitomi,Uchiyama, Harue,Ohura, Takeshi,Sakakibara, Hiroyuki,Kuruto, Ryoko,Amagai, Takashi,Shimoi, Kayoko
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experimental part
p. 70 - 76
(2011/02/22)
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- An expedient one-pot entry to catecholestrogens and other catechol compounds via IBX-mediated phenolic oxygenation
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A one-pot procedure for the preparation of catecholestrogens in over 90% yield is reported, involving oxygenation of 17β-estradiol or estrone with o-iodoxybenzoic acid (IBX) followed by reduction with methanolic NaBH 4. The procedure, which was extended to the o-hydroxylation of a number of representative phenols in good-to-high yields, expands significantly the scope of phenolic oxidation mediated by IBX.
- Pezzella, Alessandro,Lista, Liliana,Napolitano, Alessandra,D'Ischia, Marco
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p. 3541 - 3544
(2007/10/03)
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- Synthesis of the catechols of natural and synthetic estrogens by using 2-iodoxybenzoic acid (IBX) as the oxidizing agent
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A method for the synthesis of 2-hydroxyestrone/estradiol, 4-hydroxyestrone/estradiol, 3′-hydroxydiethylstilbestrol, 3′-hydroxyhexestrol, and 3′-hydroxydienestrol is reported, in which 2-iodoxybenzoic acid (IBX) and the corresponding phenolic estrogen are reacted. Treatment of the natural estrogens, estrone/estradiol, with stoichiometric amounts of IBX in dimethylformamide initially yielded a mixture of estrone/estradiol-2,3- and -3,4-quinones, which were reduced in situ to the corresponding catechols by treatment with a 1 M aqueous solution of ascorbic acid. Chromatographic separation of the reaction products afforded 2- and 4-hydroxyestrone/estradiol in good overall yields (79%). In the case of the synthetic estrogens containing two identical phenolic rings, protection of one ring is a prerequisite for the synthesis of the monocatechol. Thus, diethylstilbestrol and dienestrol were protected at one phenol ring as their methyl ethers. The resulting monophenols were treated with stoichiometric amounts of IBX for 1 h, followed by treatment with 1 M aqueous ascorbic acid to obtain the corresponding catechols in more than 70% yield. Furthermore, the catechol of diethylstilbestrol, protected at one ring, was reduced by catalytic hydrogenation at the C3-C4 double bond to obtain 3′-hydroxyhexestrol in 90% yield. Removal of the protected methoxy groups of the synthetic estrogen catechols was carried out by treatment with a 1 M solution of boron tribromide in dichloromethane. This method is highly efficient for the preparative scale synthesis of catechols of both natural and synthetic estrogens.
- Saeed, Muhammad,Zahid, Muhammad,Rogan, Eleanor,Cavalieri, Ercole
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p. 173 - 178
(2007/10/03)
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- Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes
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Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16α-position but was less active in estrone 16α-hydroxylation; the latter reaction and also estradiol 16α-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16α-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16α-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. α- Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, α-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis.
- Yamazaki, Hiroshi,Shaw, Peter M.,Guengerich, F. Peter,Shimada, Tsutomu
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p. 659 - 665
(2007/10/03)
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- CYCLODEXTRIN-FACILITATED BIOCONVERSION OF 17β-ESTRADIOL BY A PHENOLOXIDASE FROM MUCUNA PRURIENS CELL CULTURES
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After complexation with β-cyclodextrin, the phenolic steroid 17β-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens.By complexation with β-cyclodextrin the solubility of the steroid increased from almost insoluble to 660μM.The bioconversion efficiency after 72 hr increased in the following order: freely suspended cells (0percent), immobilized cells (1percent), cell homogenate (6percent), phenoloxidase preparation (40percent).Mushroom tyrosinase converted 17β-estradiol, as a complex with β-cyclodextrin, solely into 2-hydroxyestradiol, with a maximal yield of 30percentafter 6-8 hr.Uncomplexed 17β-estradiol was not converted at all in any of these systems.
- Woerdenbag, Herman J.,Pras, Niesko,Frijlink, Henderik W.,Lerk, Coenraad F.,Malingre, Theo M.
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p. 1551 - 1554
(2007/10/02)
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