Dietary supplements containing black cohosh are alternatives to conventional hormone replacement therapy in menopause. This study investigates the maximum tolerated dose of a 75% ethanol extract of black cohosh and determines the pharmacokinetics of one of its most abundant triterpene glycosides, 23-epi-26-deoxyactein. Single doses of black cohosh extract containing 1.4, 2.8, or 5.6 mg of 23-epi-26-deoxyactein were administered to 15 healthy, menopausal women. Serial blood samples and 24-h urine samples were obtained; blood chemistry, hormonal levels, and 23-epi-26-deoxyactein levels were determined. No acute toxicity or estrogenic hormone effects were observed. Pharmacokinetic analyses of 23-epi-26-deoxyactein in sera indicated that the maximum concentration and area under the curve increased proportionately with dosage, and that the half-life was ~2 h for all dosages. Less than 0.01% of the 23-epi-26-deoxyactein was recovered in urine 24 h after administration. No phase I or phase II metabolites were observed either in clinical specimens or in vitro.

Estrogen exposure is a risk factor for breast cancer and estrogen oxidative metabolites have been implicated in chemical carcinogenesis. Oxidation of the catechol metabolite of estrone (4-OHE) and the beta-naphthohydroquinone metabolite of equilenin (4-OHEN) gives o-quinones that produce ROS and damage DNA by adduction and oxidation. To differentiate hormonal and chemical carcinogensis pathways in estrogen receptor positive ER(+) cells, catechol or beta-naphthohydroquinone warheads were conjugated to the selective estrogen receptor modulator (SERM) desmethylarzoxifene (DMA). ER binding was retained in the DMA conjugates; both were antiestrogens with submicromolar potency in mammary and endometrial cells. Cytotoxicity, apoptosis, and caspase-3/7 activation were compared in ER(+) and ER(-)MDA-MB-231 cells, and production of ROS was detected using a fluorescent reporter. Comparison was made to DMA, isolated warheads, and a DMA-mustard. Conjugation of warheads to DMA increased cytotoxicity accompanied by induction of apoptosis and activation of caspase-3/7. Activation of caspase-3/7, induction of apoptosis, and cytotoxicity were all increased significantly in ER(+) cells for the DMA conjugates. ROS production was localized in the nucleus for conjugates in ER(+) cells. Observations are compatible with beta-naphthohydroquinone and catechol groups being concentrated in the nucleus by ER binding, where oxidation and ROS production result, concomitant with caspase-dependent apoptosis. The results suggest DNA damage induced by catechol estrogen metabolites can be amplified in ER(+) cells independent of hormonal activity. The novel conjugation of quinone warheads to an ER-targeting SERM gives ER-dependent, enhanced apoptosis in mammary cancer cells of potential application in cancer therapy.

"Southern Resident" killer whales (Orcinus orca) that comprise three fish-eating "pods"(J, K and L) were listed as "endangered" in the US and Canada following a 20% population decline between 1996 and 2001. Blubber biopsy samples from Southern Resident juveniles had statistically higher concentrations of certain persistent organic pollutants than were found for adults. Most Southern Resident killer whales, including the four juveniles, exceeded the health-effects threshold for total PCBs in marine mammal blubber. Maternal transfer of contaminants to the juveniles during rapid development of their biological systems may put these young whales at greater risk than adults for adverse health effects (e.g., immune and endocrine system dysfunction). Pollutant ratios and field observations established that two of the pods (K- and L-pod) travel to California to forage. Nitrogen stable isotope values, supported by field observations, indicated possible changes in the diet of L-pod over the last decade.

The equine estrogens equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive <i>o</i>-quinone form in aerated aqueous solutions. The <i>o</i>-quinones react predominantly with C, and to a lesser extent with A and G, to form pre-mutagenic cyclic covalent DNA adducts in vitro and in vivo. To obtain insights into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically modified oligonucleotides containing the stereoisomeric <i>1'S,2'R,3'R</i>-4-OHEN-C3 and <i>1'R,2'S,3'S</i>-4-OHEN-C4 adducts derived from the reaction of 4-OHEN with the C in the oligonucleotide 5'-GGTAGCGATGG in aqueous solution. A combined NMR and computational approach was utilized to determine the conformational characteristics of the two major 4-OHEN-C3 and 4-OHEN-C4 stereoisomeric adducts formed in this oligonucleotide hybridized with its complementary strand. In both cases, the modified C adopts an <i>anti</i> glycosidic bond conformation; the equilenin distal ring protrudes into the minor groove while its two proximal hydroxyl groups are exposed on the major groove side of the DNA duplex. The bulky 4-OHEN-C adduct distorts the duplex within the central GC*G portion, but Watson-Crick pairing is maintained adjacent to C* in both stereoisomeric adducts. For the 4-OHEN-C3 adduct, the equilenin rings are oriented toward the 5'-end of the modified strand, while in 4-OHEN-C4 the equilenin is 3'-directed. Correspondingly, the distortions of the double-helical structures are more pronounced on the 5'-, or the 3'-side of the lesion, respectively. These differences in stereoisomeric adduct conformations may play a role in the processing of these lesions in cellular environments.

Estrogen-DNA adducts are potential biomarkers for assessing cancer risk and progression in estrogen-dependent cancer. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in hormone replacement therapy formulations, autoxidizes to a reactive o-quinone that subsequently causes DNA damage. The formation of stable stereoisomeric cyclic 4-OHEN-DNA adducts has been reported in vitro and in vivo, but their removal by DNA repair processes in cells has not been determined. Such studies have been hampered by low yields of cyclic adducts and poor reproducibility when treating cells in culture with 4-OHEN. These problems are attributed in part to the instability of 4-OHEN in aerobic, aqueous media. We show herein that low yields and reproducibility can be overcome by 4-OHEN diacetate as a novel, cell-permeable 4-OHEN precursor, in combination with a sensitive LC-MS/MS method developed for detecting adducts in human breast cancer cells. This method involves isolation of cellular DNA, DNA digestion to deoxynucleosides, followed by the addition of an isotope-labeled internal standard (4-OHEN-(15)N(5)-dG adduct) prior to analysis by LC-MS/MS. A concentration-dependent increase in adduct levels was observed in MCF-7 cells after exposure to 4-OHEN diacetate. The chemical stabilities of the adducts were also investigated to confirm that adducts were stable under assay conditions. In conclusion, this newly developed LC-MS/MS method allows detection and relative quantification of 4-OHEN-DNA adducts in human breast cancer cells, which could be adapted for adduct detection in human samples.

Conjugation of biotin and fluorophore tags is useful for assaying covalent protein modification. Oxidative bioactivation of selective estrogen receptor modulators (SERMs) yields reactive quinoid electrophiles that covalently modify proteins, and bioactivation is associated with carcinogenic and chemopreventive effects. Identification of the protein targets of electrophilic metabolites is of general importance for xenobiotics. Four methodologies using SERM derivatized biotin/fluorophore tags were compared for purification and quantification:(1) covert oxidatively activated tags (COATags; SERM conjugated to biotin);(2) dansylTags (SERM conjugated to fluorophore); and azidoTags (SERM azide derivatives) in a two-step conjugation to biotin, using either (3) Staudinger ligation or (4) click chemistry. All synthetic derivatives retained the estrogen receptor ligand characteristics of the parent SERMs. Model proteins with bioactivation by tyrosinase in buffer or cell lysates and liver proteins with in situ bioactivation in rat primary hepatocytes were studied by immunoassay and fluorescence. Comparison showed that the azidoTag/Staudinger method was sensitive but nonspecific, the azidoTag/click methodology had low sensitivity, and the dansylTag methodology failed to detect modified proteins in hepatocytes. The COATag methodology was judged superior, detecting 5 ng of modified protein in vitro and identifying protein targets in hepatocytes. In metabolism studies in rat liver microsomes, the azide group was metabolically labile, which was a contributing factor in not selecting the azidoTag methodology in the oxidative environments required for bioactivation. For study of the protein targets of electrophilic metabolites formed by in situ oxidative bioactivation, the COATag is both sensitive and specific and does not appear to suffer from poor cell permeability.

Teleost embryos develop a syndrome characterized by edema when exposed to water that weathers substrates contaminated with crude oil. Previous studies using zebrafish demonstrated that crude oil exposure causes cardiogenic edema, and that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils (tricyclic fluorenes, dibenzothiophenes, and phenanthrenes) are cardiotoxic, causing arrhythmia through a pathway that does not require activation of the aryl hydrocarbon receptor (AHR). We demonstrate here for Pacific herring, a species impacted by the Exxon Valdez oil spill, that the developing heart is the primary target of crude oil exposure. Herring embryos exposed to the effluent of oiled gravel columns developed dose-dependent edema and irregular cardiac arrhythmia soon afterthe heartbeat was established. At a dose that produced cardiac dysfunction in 100% of exposed embryos, tissue levels of tricyclic PAHs were below 1 micromol/kg, suggesting a specific, high affinity target in the heart. These findings have implications for understanding the mechanism of tricyclic PAH cardiotoxicity, the development of biomarkers for the effects of PAH exposure in fish, and understanding the long-term impacts of oil spills and other sources of PAH pollution in aquatic environments.

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 (ChIP) assay was found to activate ERa binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells, comparing ERa(+) versus ERa(-) cells, and comparing 4-OHEN versus menadione, a reactive oxygen species (ROS) generating, but non-estrogenic quinone. 4-OHEN selectively induced DNA damage in ERa(+) cells, whilst menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERa(+) 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 ERa(+) cells within 5 min, whereas in ERa(-) cells or menadione-treated cells, no selectivity was observed. These data support ERa 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.

INTRODUCTION: Earlier studies reported serotonergic activity for cimicifugic acids (CA) isolated from Cimicifuga racemosa. The discovery of strongly basic alkaloids, cimipronidines, from the active extract partition and evaluation of previously employed work-up procedures has led to the hypothesis of strong acid/base association in the extract. OBJECTIVE: Re-isolation of the CAs was desired to permit further detailed studies. Based on the acid/base association hypothesis, a new separation scheme of the active partition was required, which separates acids from associated bases. METHODOLOGY: A new 5-HT(7) bioassay guided work-up procedure was developed that concentrates activity into one partition. The latter was subjected to a new two-step centrifugal partitioning chromatography (CPC) method, which applies pH zone refinement gradient (pHZR CPC) to dissociate the acid/base complexes. The resulting CA fraction was subjected to a second CPC step. Fractions and compounds were monitored by (1)H NMR using a structure-based spin-pattern analysis facilitating dereplication of the known acids. Bioassay results were obtained for the pHZR CPC fractions and for purified CAs. RESULTS: A new CA was characterised. While none of the pure CAs was active, the serotonergic activity was concentrated in a single pHZR CPC fraction, which was subsequently shown to contain low levels of the potent 5-HT(7) ligand, N(omega)-methylserotonin. CONCLUSION: This study shows that CAs are not responsible for serotonergic activity in black cohosh. New phytochemical methodology (pHZR CPC) and a sensitive dereplication method (LC-MS) led to the identification of N(omega)-methylserotonin as serotonergic active principle. Copyright (c) 2009 John Wiley & Sons, Ltd.