JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.. The National Institute of Environmental Health Sciences (NIEHS) and Brogan & Partners are collaborating with JSTOR to digitize, preserve and extend access to Environmental Health Perspectives.Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5,7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4',5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4'-methoxyisoflavone)], one flavanone [naringenin (4',5,7-trihydroxyflavanone)], and one naphthoflavone (a-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are signifirntly poorer inhibitors of aromatase than flavones. Key words: aromatase, aromatase inhibitors, computer modeling, cytochrome P450, estrogen synthesis, flavone, isoflavone, phytoestrogens, site-directed mutagenesis. Environ Health Perspect 106:85-92 (1998). [Online 21 January 1998] http. //ehpnetlniehs.nih.gov/docs/1998/106p85-92kao/abstract.html As pointed out by Henderson (1) in 1993, one of the most dramatic features of breast cancer is the disparity in incidence rates between highly westernized and nonwesternized countries. Women born and raised in the UnitedStates are at least five times as likely to get breast cancer as women born and raised in Japan. But Japanese women increase their risk if they live in rapidly westernizing Japanese cities or emigrate and live in the United States. As is true with coronary heart disease, differences in diet are thought to be a major underlying factor in the different incidence rates of breast cancer, particularly among postmenopausal women. The U.S. Environmental Protection Agency (EPA) anticipates filling at least one position in the Office of Economy and Environment. The individual will conduct original research on environmental health issues. Individuals with an interest in conducting policyrelevant an...

Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones.ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6

Estrogen is a major factor in the development of breast cancer. In situ estrogen production by aromatase/estrogen synthetase in breast cancer plays a dominant role in tumor proliferation. Because natural compounds such as flavones and isoflavones have been shown to be inhibitors of aromatase, it is thought that vegetables that contain these phytochemicals can inhibit aromatase activity and suppress breast cancer cell proliferation. Heat-stable extracts were prepared from vegetables and screened for their ability to inhibit aromatase activity in a human placental microsome assay. The white button mushroom (species Agaricus bisporus) suppressed aromatase activity dose dependently. Enzyme kinetics demonstrated mixed inhibition, suggesting the presence of multiple inhibitors or more than one inhibitory mechanism. "In cell" aromatase activity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phytochemicals in the mushroom aqueous extract inhibited aromatase activity and proliferation of MCF-7aro cells. These results suggest that diets high in mushrooms may modulate the aromatase activity and function in chemoprevention in postmenopausal women by reducing the in situ production of estrogen.

Aromatase (CYP19) catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. In this study, five human aromatase mutants (E302D, S478A, S478T, H480K, and H480Q) were prepared using a mammalian cell expression system. These mutants were evaluated by enzyme kinetic analysis, inhibitory profile studies, and reaction intermediate measurements. Three steroidal inhibitors [4-hydroxyandrostenedione (4-OHA), 7a-(4 H -amino)phenylthio-1,4-androstandiene-3,17-dione (7a-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)] were used in the inhibitory profile studies. Our computer model of aromatase suggests that Glu302 is situated in the conserved I-helix region and located near the C-19 position of the steroid substrate. The model was supported by significant changes in kinetic parameters and a sevenfold increase in the K i value of MDL 101,003 for the mutant E302D. As S478A was found to have kinetic properties similar to the wild-type enzyme and a much higher activity than S478T, Ser478 is thought to be situated in a rather restricted environment. There was a 10-fold increase in the K i value of 7a-APTADD for S478T over that for the wild-type enzyme, suggesting that Ser478 might be near the C-7 position of the substrate. The reaction intermediate analysis revealed that significantly more 19-ol intermediate was generated by both S478A and S478T than the wild-type enzyme. These results would support a hypothesis that Ser478 plays a role in the first and second hydroxylation reactions. A positive charged amino acid is preferred at position 480 as shown by the fact that H480K has a significantly higher activity than H480Q. The K i value of 4-OHA for H480Q was found to be three times that of the wild-type enzyme. In addition, significantly more 19-ol and 19-al intermediates were detected for both mutants H480K and H480Q than for the wild-type enzyme. Evaluation of the two mutations at His480 allows us to propose that this residue may participate in the aromatization reaction (the third step) by acting as a hydrogen bond donor for the C-3 keto group of the substrate. Furthermore, new products were generated when the enzyme was mutated at Ser478 and His480. Thus, these two residues must play an important role in the catalysis and are likely closer to the substrate binding site than previously predicted.

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