Cytochrome P450 enzymes that metabolize estrogens are expressed in the mammary gland, uterus, brain and other target tissues for estrogen action, and this results in the formation of hydroxylated estrogens in these tissues. Estradiol metabolites formed in target tissues at or near estrogen receptors may either be inactive or have important biological effects, and changes in the activities of estrogen-metabolizing enzymes in target tissues may profoundly influence estrogen action. Although some active estrogen metabolites exert hormonal effects in target tissues by interaction with the classical estrogen receptor, other metabolites appear to elicit unique biological responses that are not associated with activation of this receptor. Therefore, some of the many actions of estradiol may not be caused by estradiol per se, but may result from the formation of active estrogen metabolite(s) which function as local mediators or may activate their own unique receptors or effectors. This is an important area in need of more research. The present paper represents a review of the literature and perspectives by the authors on the functional role of estrogen metabolism in target tissues.

In the present investigation, we studied the modulating effects of several tea catechins and bioflavonoids on DNA methylation catalyzed by prokaryotic SssI DNA methyltransferase (DNMT) and human DNMT1. We found that each of the tea polyphenols [catechin, epicatechin, and (Ϫ)-epigallocatechin-3-O-gallate (EGCG)] and bioflavonoids (quercetin, fisetin, and myricetin) inhibited SssI DNMT-and DNMT1-mediated DNA methylation in a concentration-dependent manner. The IC 50 values for catechin, epicatechin, and various flavonoids ranged from 1.0 to 8.4 M, but EGCG was a more potent inhibitor, with IC 50 values ranging from 0.21 to 0.47 M. When epicatechin was used as a model inhibitor, kinetic analyses showed that this catecholcontaining dietary polyphenol inhibited enzymatic DNA methylation in vitro largely by increasing the formation of S-adenosyl-L-homocysteine (a potent noncompetitive inhibitor of DNMTs) during the catechol-O-methyltransferase-mediated Omethylation of this dietary catechol. In comparison, the strong inhibitory effect of EGCG on DNMT-mediated DNA methylation was independent of its own methylation and was largely due to its direct inhibition of the DNMTs. This inhibition is strongly enhanced by Mg 2ϩ . Computational modeling studies showed that the gallic acid moiety of EGCG plays a crucial role in its high-affinity, direct inhibitory interaction with the catalytic site of the human DNMT1, and its binding with the enzyme is stabilized by Mg 2ϩ . The modeling data on the precise molecular mode of EGCG's inhibitory interaction with human DNMT1 agrees perfectly with our experimental finding.DNA methylation at the C5-position of cytosine within the CpG dinucleotides represents an important mechanism for epigenetic control of gene expression and the maintenance of genome integrity. Although DNA hypermethylation is associated with inactivation of genes, global genomic hypomethylation is often associated with the induction of chromosomal instability (Chen et al., 1998). A number of recent studies have suggested that bioactive food components, including both essential and nonessential nutrients, can modify DNA methylation patterns in complex ways. For instance, consumption of a methyl-deficient diet led to hypomethylation of specific CpG sites within several oncogenes (such as c-myc, c-fos, and c-H-ras), resulting in elevated levels of mRNA for these genes (Wainfan and Poirier, 1992). A recent study showed that tea catechins are effective inhibitors of human DNA methyltransferase (DNMT)-mediated DNA methylation in vitro and in cultured cancer cells (Fang et al., 2003). DNA methylation is catalyzed by specific DNMTs. Multiple DNMTs are known to be present in humans, animals, and microorganisms, and they have varying degrees of specificity toward unmethylated and hemimethylated DNA substrates (Bestor and Ingram, 1983). S-Adenosyl-L-methionine (SAM) is the methyl donor in the DNMT-mediated DNA methylation, as in many other enzymatic methylation reactions [such as the catechol-O-methyltransferase (COMT)-med...

We systematically characterized the oxidative metabolites of 17beta-estradiol and estrone formed by 15 human cytochrome P450 (CYP) isoforms. CYP1A1 had high activity for 17beta-estradiol 2-hydroxylation, followed by 15alpha-, 6alpha-, 4-, and 7alpha-hydroxylation. However, when estrone was the substrate, CYP1A1 formed more 4-hydroxyestrone than 15alpha- or 6alpha-hydroxyestrone, with 2-hydroxyestrone as the major metabolite. CYP1A2 had the highest activity for the 2-hydroxylation of both 17beta-estradiol and estrone, although it also had considerable activity for their 4-hydroxylation (9-13% of 2-hydroxylation). CYP1B1 mainly catalyzed the formation of catechol estrogens, with 4-hydroxyestrogens predominant. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2D6 each showed a varying degree of low catalytic activity for estrogen 2-hydroxylation, whereas CYP2C18 and CYP2E1 did not show any detectable estrogen-hydroxylating activity. CYP3A4 had strong activity for the formation of 2-hydroxyestradiol, followed by 4-hydroxyestradiol and an unknown polar metabolite, and small amounts of 16alpha- and 16beta-hydroxyestrogens were also formed. The ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone with CYP3A4 was 0.22 or 0.51, respectively. CYP3A5 had similar catalytic activity for the formation of 2- and 4- hydroxyestrogens. Notably, CYP3A5 had an unusually high ratio of 4- to 2-hydroxylation of 17beta-estradiol or estrone (0.53 or 1.26, respectively). CYP3A4 and 3A5 also catalyzed the formation of nonpolar estrogen metabolite peaks (chromatographically less polar than estrone). CYP3A7 had a distinct catalytic activity for the 16alpha-hydroxylation of estrone, but not 17beta-estradiol. CYP4A11 had little catalytic activity for the metabolism of 17beta-estradiol and estrone. In conclusion, many human CYP isoforms are involved in the oxidative metabolism of 17beta-estradiol and estrone, with a varying degree of catalytic activity and distinct regioselectivity.

We studied the modulating effects of caffeic acid and chlorogenic acid (two common coffee polyphenols) on the in vitro methylation of synthetic DNA substrates and also on the methylation status of the promoter region of a representative gene in two human cancer cells lines. Under conditions that were suitable for the in vitro enzymatic methylation of DNA and dietary catechols, we found that the presence of caffeic acid or chlorogenic acid inhibited in a concentration-dependent manner the DNA methylation catalyzed by prokaryotic M.SssI DNA methyltransferase (DNMT) and human DNMT1. The IC50 values of caffeic acid and chlorogenic acid were 3.0 and 0.75 microM, respectively, for the inhibition of M.SssI DNMT-mediated DNA methylation, and were 2.3 and 0.9 microM, respectively, for the inhibition of human DNMT1-mediated DNA methylation. The maximal in vitro inhibition of DNA methylation was approximately 80% when the highest concentration (20 microM) of caffeic acid or chlorogenic acid was tested. Kinetic analyses showed that DNA methylation catalyzed by M.SssI DNMT or human DNMT1 followed the Michaelis-Menten curve patterns. The presence of caffeic acid or chlorogenic acid inhibited DNA methylation predominantly through a non-competitive mechanism, and this inhibition was largely due to the increased formation of S-adenosyl-L-homocysteine (SAH, a potent inhibitor of DNA methylation), resulting from the catechol-O-methyltransferase (COMT)-mediated O-methylation of these dietary catechols. Using cultured MCF-7 and MAD-MB-231 human breast cancer cells, we also demonstrated that treatment of these cells with caffeic acid or chlorogenic acid partially inhibited the methylation of the promoter region of the RARbeta gene. The findings of our present study provide a general mechanistic basis for the notion that a variety of dietary catechols can function as inhibitors of DNA methylation through increased formation of SAH during the COMT-mediated O-methylation of these dietary chemicals.

To search for endogenous estrogens that may have preferential binding affinity for human estrogen receptor (ER) alpha or beta subtype and also to gain insights into the structural determinants favoring differential subtype binding, we studied the binding affinities of 74 natural or synthetic estrogens, including more than 50 steroidal analogs of estradiol-17beta (E2) and estrone (E1) for human ER alpha and ER beta. Many of the endogenous estrogen metabolites retained varying degrees of similar binding affinity for ER alpha and ER beta, but some of them retained differential binding affinity for the two subtypes. For instance, several of the D-ring metabolites, such as 16 alpha-hydroxyestradiol (estriol), 16 beta-hydroxyestradiol-17 alpha, and 16-ketoestrone, had distinct preferential binding affinity for human ER beta over ER alpha (difference up to 18-fold). Notably, although E2 has nearly the highest and equal binding affinity for ER alpha and ER beta, E1 and 2-hydroxyestrone (two quantitatively predominant endogenous estrogens in nonpregnant woman) have preferential binding affinity for ER alpha over ER beta, whereas 16 alpha-hydroxyestradiol (estriol) and other D-ring metabolites (quantitatively predominant endogenous estrogens formed during pregnancy) have preferential binding affinity for ER beta over ER alpha. Hence, facile metabolic conversion of parent hormone E2 to various metabolites under different physiological conditions may serve unique functions by providing differential activation of the ER alpha or ER beta signaling system. Lastly, our computational three-dimensional quantitative structure-activity relationship/comparative molecular field analysis of 47 steroidal estrogen analogs for human ER alpha and ER beta yielded useful information on the structural features that determine the preferential activation of the ER alpha and ER beta subtypes, which may aid in the rational design of selective ligands for each human ER subtype.