Scope Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue specific activity have not been analyzed. Methods and results A standardized hop extract (p.o.) and XH (s.c.) were administered to Sprague-Dawley rats over four days. LC-MS-MS analysis of plasma, liver and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, 1) XH modified Keap1 leading to Nrf2 translocation and antioxidant response element (ARE) activation; 2) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; 3) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to ERα, recruiting Nrf2, and downregulating ARE-regulated genes. Conclusions XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.

Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes: from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures, however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological “NO carrier”, GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O2 and cysteine, although, all NO-donors were oxidants towards GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method, therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible not only to quantitate S-nitrosation of GST-P1, but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1), moreover d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols.

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ER␣ binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ER␣(؉) versus ER␣(؊) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ER␣(؉) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ER␣(؉) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ER␣(؉) cells within 5 min, whereas in ER␣(؊) or menadione-treated cells, no selectivity was observed. These data support ER␣ acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.An increased relative risk of breast cancer in postmenopausal women is strongly linked to several endocrine-related risk factors. One of these risk factors is long-term exposure to hormone or estrogen replacement therapy (HRT 3 or ERT). The most widely prescribed formulations in the United States contain conjugated human estrogens and B-ring unsaturated conjugated equine estrogens, the latter constituting approximately half of the estrogen content of these formulations (1). Observations from various clinical trials and epidemiological studies collectively support the hypothesis that estrogen contributes to breast cancer and is probably causative (2-8). The large prospective Women's Health Initiative Study comparing postmenopausal women assigned HRT/ERT or placebo was terminated because of significant increases in breast cancer, stroke, and pulmonary embolism associated with therapy (9, 10). A recent analysis of data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) registries showed that the age-adjusted incidence rate of breast cancer fell 6.7% in 2003 compared with 2002, which was linked to lowered use of HRT/ERT (11). The collective evidence supports contributions to estrogensensitive breast cancer from both the proliferative and antiapoptotic hormonal effects of estrogen itself (12-16) and the genotoxic and mutagenic effects of estrogen metabolites (...

Although group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) has been reported to be phosphorylated at multiple Ser residues, the mechanisms by which phosphorylation at different sites regulates cPLA 2 ␣ activities are not fully understood. To explore the possibility that phosphorylation of Ser 727 modulates cellular protein-protein interactions, we measured the effect of Ser 727 mutations on the interaction of cPLA 2 ␣ with a reported cPLA 2 ␣-binding protein, p11. In vitro activity assays and membrane binding measurements by surface plasmon resonance analysis showed that a heterotetramer (A2t) of p11 and annexin A2, but not p11 or annexin A2 alone, directly binds cPLA 2 ␣ via Ser 727 , which keeps the enzyme from binding the membrane and catalyzing the phospholipid hydrolysis. Phosphorylation of Ser 727 disrupts this inhibitory cPLA 2 ␣-A2t interaction, thereby activating cPLA 2 ␣. Subcellular translocation and activity measurements in HEK293 cells cotransfected with cPLA 2 ␣ and p11 also showed that p11, in the form of A2t, inhibits cPLA 2 ␣ by the same mechanism and that phosphorylation of Ser 727 activates cPLA 2 ␣ by interfering with the inhibitory cPLA 2 ␣-A2t interaction. Collectively, these studies provide new insight into the regulatory mechanism of cPLA 2 ␣ through Ser 727 phosphorylation.

The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA), includes induction of chemopreventive mechanisms, and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the “NO-specific” 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM-donor, bioactivated by cellular esterase activity to release salicylates, NO3−, and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.