Gut microbial β-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. Furthermore, bacterial GUS enzymes within the gastrointestinal tract have been postulated to be a contributing factor in hormone-driven cancers. However, to date, there has been no experimental evidence to support these hypotheses. Here we provide the first in vitro analysis of the ability of 35 human gut microbial GUS enzymes to reactivate two distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide, to estrone and estradiol, respectively. We show that certain members within the Loop 1, mini-Loop 1, and FMN-binding classes of gut microbial GUS enzymes can reactivate estrogens from their inactive glucuronides. We provide molecular details of key interactions that facilitate these catalytic processes and present the structures of two novel human gut microbial GUS enzymes related to the estrobolome. Further, we demonstrate that estrogen reactivation by Loop 1 bacterial GUS enzymes can be inhibited both in purified enzymes and in fecal preparations of mixed murine fecal microbiota. Finally, however, despite these in vitro and ex vivo data, we show that a Loop 1 GUS-specific inhibitor is not capable of reducing the development of tumors in the PyMT mouse model of breast cancer. These findings validate that gut microbial GUS enzymes participate in the estrobolome but also suggest that the estrobolome is a multidimensional set of processes on-going within the mammalian gastrointestinal tract that likely involves many enzymes, including several distinct types of GUS proteins.

Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo including, acute cytotoxicity, immunotoxicity, and carcinogenesis. In contrast, quinones can induce cytoprotection through the induction of detoxification enzymes, anti-inflammatory activities, and modification of redox status. The mechanisms by which quinones cause these effects can be quite complex. The various biological targets of quinones depend on their rate and site of formation and their reactivity. Quinones are formed through a variety of mechanisms from simple oxidation of catechols/hydroquinones catalyzed by a variety of oxidative enzymes and metal ions to more complex mechanisms involving initial P450-catalyzed hydroxylation reactions followed by two-electron oxidation. Quinones are Michael acceptors, and modification of cellular processes could occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radical anions leading to the formation of reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can alter redox balance within cells through the formation of oxidized cellular macromolecules including lipids, proteins, and DNA. This perspective explores the varied biological targets of quinones including GSH, NADPH, protein sulfhydryls [heat shock proteins, P450s, cyclooxygenase-2 (COX-2), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1, (NQO1), kelch-like ECH-associated protein 1 (Keap1), IκB kinase (IKK), and arylhydrocarbon receptor (AhR)], and DNA. The evidence strongly suggests that the numerous mechanisms of quinone modulations (i.e., alkylation versus oxidative stress) can be correlated with the known pathology/cytoprotection of the parent compound(s) that is best described by an inverse U-shaped dose-response curve.

Alcohol consumption is associated with an increased risk of breast cancer, increasing linearly even with a moderate consumption and irrespectively of the type of alcoholic beverage. It shows no dependency from other risk factors like menopausal status, oral contraceptives, hormone replacement therapy, or genetic history of breast cancer. The precise mechanism for the effect of drinking alcohol in mammary cancer promotion is still far from being established. Studies by our laboratory suggest that acetaldehyde produced in situ and accumulated in mammary tissue because of poor detoxicating mechanisms might play a role in mutational and promotional events. Additional studies indicated the production of reactive oxygen species accompanied of decreases in vitamin E and GSH contents and of glutathione transferase activity. The resulting oxidative stress might also play a relevant role in several stages of the carcinogenic process. There are reported in literature studies showing that plasmatic levels of estrogens significantly increased after alcohol drinking and that the breast cancer risk is higher in receptor ER-positive individuals. Estrogens are known that they may produce breast cancer by actions on ER and also as chemical carcinogens, as a consequence of their oxidation leading to reactive metabolites. In this review we introduce our working hypothesis integrating the acetaldehyde and the oxidative stress effects with those involving increased estrogen levels. We also analyze potential preventive actions that might be accessible. There remains the fact that alcohol drinking is just one of the avoidable causes of breast cancer and that, at present, the suggested acceptable dose for prevention of this risk is of one drink per day.

To determine whether dietary berries and ellagic acid prevent 17beta-estradiol (E(2))-induced mammary tumors by altering estrogen metabolism, we randomized August-Copenhagen Irish rats (n = 6 per group) into five groups: sham implant + control diet, E(2) implant + control diet (E(2)-CD), E(2) + 2.5% black raspberry (E(2)-BRB), E(2) + 2.5% blueberry (E(2)-BB), and E(2) + 400 ppm ellagic acid (E(2)-EA). Animals were euthanized at early (6 wk), intermediate (18 wk), and late (24 wk) phases of E(2) carcinogenesis, and the mammary tissue was analyzed for gene expression changes using quantitative real-time PCR. At 6 weeks, E(2) treatment caused a 48-fold increase in cytochrome P450 1A1 (CYP1A1; P < 0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (P < 0.001). E(2) did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively, from baseline (P < 0.05). There was a 5-fold increase in 17beta-hydroxysteroid dehydrogenase 7 (17betaHSD7), and this was moderately abrogated to approximately 2-fold by all supplementation (P < 0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E(2)-CD and only E(2)-BB reduced this increase to 7-fold (P < 0.05). Catechol-O-methyltransferase expression was elevated 2-fold by E(2) treatment (P < 0.05), and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced but still high (8-fold) in E(2)-treated rats. This increase was reduced to 3.2- and 4.6-fold by E(2)-BRB and E(2)-EA, respectively (P < 0.05), but not by E(2)-BB. Supplementation did not alter the effect of E(2) on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10-30%), volume (41-67%), and multiplicity (38-51%; P < 0.05). Berries may prevent mammary tumors by suppressing the levels of E(2)-metabolizing enzymes during the early phase of E(2) carcinogenesis.

Members of the multidrug resistance protein (MRP/ABCC) subfamily of ATP-binding cassette proteins transport a wide array of anionic compounds, including sulfate, glucuronide, and glutathione (GSH) conjugates. The present study tested the ATP-dependent vesicular transport of leukotriene C(4) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) mediated by the MRP1 and MRP2 transporters in the presence of six potential modulators from three different classes of GSH-conjugated catechol metabolites: the ecstasy metabolite 5-(glutathion-S-yl)-N-methyl-alpha-methyldopamine (5-GS-N-Me-alpha-MeDA), the caffeic acid metabolite 2-(glutathion-S-yl)-caffeic acid (2-GS-CA), and four GSH conjugates of 2-hydroxy (OH) and 4-OH estrogens (GS estrogens). MRP1-mediated E(2)17betaG transport was inhibited in a competitive manner with a relative order of potency of GS estrogens (IC(50) <1 microM) > 2-GS-CA (IC(50) 3 microM) > 5-GS-N-Me-alpha-MeDA (IC(50) 31 microM). MRP2-mediated transport was inhibited with a similar order of potency, except the 2-hydroxy-4-(glutathion-S-yl)-estradiol and 4-hydroxy-2-(glutathion-S-yl)-estradiol conjugates were approximately 50- and 300-fold less potent, respectively. Transport activity was unaffected by N-acetylcysteine conjugates of N-Me-alpha-MeDA and CA. The position of GSH conjugation appears important as all four GS estrogen conjugates tested were potent inhibitors of MRP1 transport, but only the 2-hydroxy-1-(glutathion-S-yl)-estradiol and 2-hydroxy-1-(glutathion-S-yl)-estrone conjugates were potent inhibitors of MRP2-mediated transport. In conclusion, we have identified three new classes of MRP1 and MRP2 modulators and demonstrated that one of these, the estrogen conjugates, shows unanticipated differences in their interactions with the two transporters.

There is a clear association between the excessive exposure to estrogens and the development of cancer in hormone-sensitive tissues (breast, endometrium). It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. One major pathway is the extensively studied hormonal pathway, by which estrogen stimulates cell proliferation through nuclear estrogen receptor (ER)-mediated signaling, thus resulting in an increased risk of genomic mutations during DNA replication. A similar "nongenomic pathway", potentially involving newly discovered membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways. This perspective is focused on a third pathway involving the metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze o-quinone formation, so that these electrophilic/redox-active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. It appears that the endogenous estrogen quinones primarily form unstable N3-adenine or N7-guanine DNA adducts, ultimately resulting in mutagenic apurinic sites. In contrast, equine estrogen quinones, formed from estrogens present in popular hormone replacement therapy prescriptions, generate a variety of DNA lesions, including bulky stable adducts, apurinic sites, DNA strand cleavage, and oxidation of DNA bases. DNA damage induced by these equine quinones is significantly increased in cells containing ERs, leading us to hypothesize a mechanism involving ER binding/alkylation by the catchol/quinone, resulting in a "Trojan horse". The "Trojan horse" carries the highly redox-active catechol to estrogen -sensitive genes, where high amounts of reactive oxygen species are generated, causing selective DNA damage. Our data further suggest that other key protein targets for estrogen o-quinones could be redox-sensitive enzymes (i.e, GST P1-1, QR). These proteins are involved in stress response cascades that are known to contribute to the regulation of cell proliferation and apoptosis. Finally, it has been shown that catechol estrogens can transform breast epithelial cells into a tumorigenic phenotype and that these transformed cells had differential gene expression of several genes involved in oxidative stress. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.