In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified ( n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

Reduction of estrogen-induced transformation of mouse mammary epithelial cells by N-acetylcysteine.

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-Catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-Acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well tolerated in clinical studies, it may be a promising candidate for breast cancer prevention.

[The neuroendocrine effects of neonatal exposure to an inhibitor of catechol-o-methyltransferase and of sex steroids]. / Neiroéndokrinnye éffekty neonatal'nogo vozdeistviia ingibitora katekhol-o-metiltransferazy i polovykh steroidov.

The role of catechol-o-methyltransferase (COMT) in functional interrelationship between testosterone (T), catechol estrogens (CE) and catecholamines (CA) during cerebral sex differentiation (CSD) was investigated in experiments on Wistar rats. Sex dimorphism in the level of CA in the rat hypothalamus was revealed on the 10th day of life. Noradrenaline concentration in male rats was significantly higher than in the female rats (p less than 0.05). It was shown that isolated CA accumulation in the hypothalamus of 10-day female rats by means of direct suppression of COMT with tropolon (300 micrograms on the 5th and 7th days of life) was insufficient for masculinization of sex cycling regulation centers. At the same time tropolon administered in a dose of 100 micrograms on the 4th-10th days of life enhanced the sterilizing effect of T administered in a dose of 25 micrograms on the 4th day of life. The development of anovulatory sterility (AS) was observed in 100% of cases. The neonatal effect of 2-hydroxyestradiol-17 beta (2-OH-E2 50 micrograms on the 5th day of life) and tropolon (300 micrograms on the 5th and 7th days of life) was ineffective with relation to AS induction indicating the absence of the inductor role of 2-OH-E2 in CSD. A conclusion is that CSD is a result of combined action of androgens, their metabolites (4-hydroxylated CE isomers) and COMT-mediated CA.

Cobalt-protoporphyrin causes prolonged inhibition of catechol estrogen synthesis by rat liver microsomes.

A single injection of cobalt-protoporphyrin (CoPP), which produces a marked and sustained decline in hepatic cytochrome P450 content, reduced the ability of male rat liver microsomes to form catechol estrogens to about 30% of control values within 1 day, as measured by the release of 3H2O from [2-3H]estradiol. Two days after treatment, the apparent Km of estrogen 2-hydroxylase for estradiol was increased, but other inhibitors of cytochrome P450 function (SKF-525A or piperonyl butoxide) failed to affect the enzyme. Inhibition by CoPP was also demonstrated by measuring the conversion of [4-14C]estradiol to its 2-hydroxylated derivative visualized by autoradiography after chromatographic separation. These findings point to yet another site in the multifaceted action of cobalt protoporphyrin.