17beta-Estradiol nitration by peroxidase/H2O2/NO2-: a chemical assessment

Nitration of 17b-estradiol by H2O2 and nitrite in the presence of various peroxidases, viz. horseradish peroxidase, lactoperoxidase, and peroxidase-containing homogenates from bovine uteri, was systematically investigated to assess on a chemical basis its potential relevance to the mechanisms of impairment of estrogen functions under oxidative/nitrosative stress conditions. In the presence of excess nitrite 17b-estradiol reacted smoothly to give 2-nitroestradiol (1), 4-nitroestradiol (2), and 2,4-dinitroestradiol (3). With 10–300 lM estradiol, formation yields of 1–3 were 12–55%, but dropped to 1% or less at lower estrogen concentration, for example, 1 lM, or in plasma as the reaction medium. Time course analysis showed that 2 is the prevalent nitration product under conditions of slow generation of nitrating species, suggesting some regioselectivity for estradiol nitration at C-4, whereas 1 prevails with bolus addition of reagents, due to faster degradation of 2. Competition experiments carried out with 15NO2 showed that 2 is about twice more susceptible to nitration than 1 as determined by 15N NMR analysis of the resulting 3. The biological effects of 1 and 2 were preliminarily tested on in vitro bovine embryo cultures. When 1 and 2 were substituted to the standard 17b-estradiol in the oocyte maturation, a significant decrease in both cleavage and blastocyst efficiency was observed in the case of 1 but not 2. Overall, these results suggest that estradiol nitration is a potential pathway of hormonal dysfunction and toxicity but would require elevated estrogen levels of questionable physiological relevance.