title: In vitro generation of peroxynitrite by 2- and 4-hydroxyestrogens in the presence of nitric oxide.
abstract: Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.
authors.affiliation: Département de Médecine Nucléaire et de Radiobiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. bpaque01@courrier.usherb.ca
authors.forename: B
authors.initials: B
authors.lastname: Paquette
authors.forename: A M
authors.initials: AM
authors.lastname: Cantin
authors.forename: S
authors.initials: S
authors.lastname: Kocsis-Bédard
authors.forename: S
authors.initials: S
authors.lastname: Barry
authors.forename: R
authors.initials: R
authors.lastname: Lemay
authors.forename: J P
authors.initials: JP
authors.lastname: Jay-Gerin
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004958:Estradiol / Q000031:analogs & derivatives* / Q000737:chemistry*; D002393:Estrogens, Catechol; D006894:Hydroxyestrones / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D009566:Nitrates / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*
chemical_list: D002393:Estrogens, Catechol; D006894:Hydroxyestrones; D009566:Nitrates; C039642:peroxynitric acid; D009569:Nitric Oxide; C026418:4-hydroxyestrone; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; C024980:2-hydroxyestrone