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Phytoestrogens are plant constituents that possess either estrogenic or antiestrogenic activity. Although their activities are weak as compared with human endogenous estrogens, the consumption of phytoestrogens may have clinically significant consequences. A number of botanicals, or the compounds contained therein, have been identified as putative estrogenic agents, but consensus in the biomedical community has been hampered by conflicting data from various in vitro and in vivo models of estrogenic activity. Phytoestrogens may serve as chemopreventive agents while at the same time being capable of promoting growth in estrogen receptor positive cancer cell lines. Furthermore, they may exert their estrogenic influence through receptor-dependent and/or receptor-independent mechanisms. These findings have led to speculation that phytoestrogen intake might be ill advised for patients at an increased risk for hormone-dependent cancers, cancer patients, or cancer survivors. This article will attempt to sort out discrepancies between various experimental models and establish whether certain herbs possess estrogenic activity. The review will focus on 5 popular botanical dietary supplements: Trifolium pratense (red clover), Cimicifuga racemosa (black cohosh), Humulus lupulus (hops), Angelica sinensis (dong quai), and Glycyrrhiza glabra (licorice). It will address their mechanisms of action, clinical evidence bases, and implications for use in cancer. [ABSTRACT FROM AUTHOR]

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.

Botanical dietary supplements, as compared with nutritional supplements or single-component pharmaceutical drugs, are typically less-refined preparations derived from bulk plant material and, as such, require a modified approach to their development, production, and evaluation. An integrated, multidisciplinary team of scientific and clinical investigators is required in order to develop high quality phytomedicines and rigorously evaluate their safety and efficacy. Research on botanicals involves unique challenges as plant source materials frequently vary in chemical content and may contain unwanted pesticides, heavy metals, contaminant plant species, or other adulterants. Ideally, a botanical formulation should be standardized, both chemically and biologically, by a combination of analytical techniques and bioassays. This combination approach provides multiple measures by which reproducible quality and efficacy of botanical supplements may be achieved, and is particularly useful for botanical products for which the active compound(s) have not yet been identified. Safety and toxicity should be evaluated during the supplement development process in both in vitro and in vivo systems. A number of liquid chromatography-mass spectrometry methods can aid in the assessment of purity, bioavailability, toxicity, metabolism, and molecular target profiling of botanical extracts. Clinical investigators must appreciate the complexity of multi-component phytomedicines and adjust trial protocols accordingly. This review highlights practical considerations of value to basic science and clinical investigators engaged in the study of botanical supplements. Lessons and examples are drawn from the authors' experience in designing and developing a red clover (Trifolium pratense L.) standardized extract for evaluation in Phase I and Phase II clinical trials.

Extracts of the rhizome of black cohosh [Actaea racemosa L., formerly called Cimicifuga racemosa (L.) Nutt.] were evaluated for potential mechanisms of action in the alleviation of menopausal hot flashes. Ovariectomized Sprague-Dawley rats were administered a 40% 2-propanol extract of black cohosh [4, 40, and 400 mg/(kg.day)] by gavage for 2 weeks with or without estradiol [50 microg/(kg.day)] to determine if black cohosh could act as an estrogen or antiestrogen on the basis of an increase in uterine weight or vaginal cellular cornification. No effects were observed on uterine weight or on vaginal cellular cornification in rats treated with black cohosh alone or in combination with 17beta-estradiol, indicating this black cohosh extract had no estrogenic or antiestrogenic properties in the ovariectomized rat model. To evaluate other potential pathways by which black cohosh might reduce menopausal hot flashes, serotonin activity was first assessed by the inhibition of radioligand binding to cell membrane preparations containing recombinant human serotonin receptor (5-HT) subtypes. A 40% 2-propanol extract of black cohosh was tested against 10 subtypes of the serotonin receptor, revealing the presence of compounds with strong binding to the 5-HT(1A), 5-HT(1D), and 5-HT(7) subtypes. Subsequent binding studies were carried out using 5-HT(1A) and 5-HT(7) receptors because of their association with the hypothalamus, which has been implicated in the generation of hot flashes. The black cohosh 40% 2-propanol extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(7) receptor (IC(50) = 2.4 +/- 0.4 microg/mL) with greater potency than binding of [(3)H]-8-hydroxy-2-(di-N-propylamino)tetralin to the rat 5-HT(1A) receptor (IC(50) = 13.9 +/- 0.6 microg/mL). Analysis of ligand binding data indicated that components of a black cohosh methanol extract functioned as a mixed competitive ligand of the 5-HT(7) receptor. In addition, a black cohosh methanol extract elevated cAMP levels in 293T-5-HT(7)-transfected HEK cells, suggesting the extract acted as a partial agonist at the receptor. The elevation in cAMP mediated by the black cohosh extract could be reversed in the presence of the antagonist methiothepin, indicating a receptor-mediated process. These data suggest that reductions in hot flashes in some women taking black cohosh may not be due to estrogenic properties. This study identifies other possible biological targets of black cohosh that could account for reported biological effects.

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins., (Copyright 2003 Wiley-Liss, Inc.)

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.

Enzymes that release 5'-deoxyribose-5-phosphate (dRP) residues from preincised apurinic/apyrimidinic (AP) DNA have been collectively termed DNA deoxyribophosphodiesterases (dRPases), but they fall into two distinct categories: the hydrolytic dRPases and AP lyases. In order to resolve a number of conflicting reports in the dRPase literature, we examined two putative hydrolytic dRPases (Escherichia coli exonuclease I (exo I) and RecJ) and four AP lyases (E. coli 2, 6-dihydroxy-5N-formamidopyrimidine (Fapy) DNA glycosylase (Fpg) and endonuclease III (endo III), bacteriophage T4 endonuclease V (endo V), and rat polymerase beta (beta-pol)) for their abilities to (i) excise dRP from preincised AP DNA and (ii) incise AP DNA. Although exo I and RecJ exhibited robust 3' to 5' and 5' to 3' exonucleolytic activities, respectively, on appropriate substrates, they failed to demonstrate detectable dRPase activity. All four AP lyases possessed both dRPase and traditional AP lyase activities, albeit to varying degrees. Moreover, as best illustrated with Fpg, AP lyase enzymes could be trapped on both preincised and unincised AP DNA using NaBH(4) as the reducing agent. These results further support the assertion that the catalytic mechanism of the AP lyases, the beta-elimination reaction, does proceed through an imine enzyme-DNA intermediate and that the active site residues responsible for dRP release must contain primary amines. Further, these data indicate a biological significance for the beta-elimination reaction of DNA glycosylase/AP lyases in that they, in concert with hydrolytic AP endonucleases, can create appropriate gapped substrates for short patch base excision repair (BER) synthesis to occur efficiently.

A recent study demonstrated that rat DNA polymerase beta (beta-pol) releases 5'-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of beta-pol and occurs via beta-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap beta-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain-DNA and intact beta-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the beta-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.

We have shown previously that 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine (125I-azido-PAPA-APEC) specifically and selectively photolabels RDC8 A2a adenosine receptors that have been overexpressed in COS M6 cells. Glycosylated, 125I-azido-PAPA-APEC-labeled, wild-type (412 residues; 45,031 Da) and carboxyl-terminally truncated (315 residues; 35,427 Da) receptors migrate with apparent molecular masses of > 40 and 31.5 kDa, respectively, whereas unglycosylated or deglycosylated wild-type and truncated A2a receptors migrate with apparent molecular masses of 40 and 28.5 kDa, respectively. Because nonspecific photoincorporation is not a complication, the present peptide mapping studies of the full length and truncated canine A2a adenosine receptors were carried out on unpurified COS M6 membrane preparations. After partial proteolysis it became clear that glycosylation increased the apparent molecular mass of either the wild-type or mutant A2a receptor by approximately 3 kDa. Although the A2a receptor was readily cleaved by a variety of chemical reagents and proteases, trypsin and endoproteinase Glu-C generated the most reproducible and, in the case of trypsin, the most complete fragmentation patterns. Radiolabeled peptides were identified by their apparent molecular masses, (in)abilities to be recognized by an antipeptide antibody to amino acids Tyr155-Val172 of the presumed second extracellular loop of the receptor, and (in)sensitivities to endoglycosidase F and tunicamycin treatments. A prominent, 7-kDa, radiolabeled peptide that was generated by trypsin digestion implicated putative alpha-helix V in the binding of 125I-azido-PAPA-APEC.

The amino acids that comprise the ligand binding sites of adenosine receptors have not been identified. Adenosine and its agonist analogues differ from ligands for the well studied biogenic amine receptors and rhodopsin in that the adenosine receptor agonists are larger, contain a ribose moiety, and are uncharged at physiological pH. Thus, the locations of the ligand binding pockets of the adenosine receptors could differ significantly from those of the biogenic amine receptors. This report describes the characterization of a purification-amenable truncated mutant of the canine A2a adenosine receptor and demonstrates that neither the long carboxyl-terminal tail nor the glycosidic moiety appears to be required for ligand binding. The dog thyroid A2a adenosine receptor cDNA (RDC8) was subcloned into the mammalian expression vector pCMV4. A mutant A2a construct, in which six histidines replaced residues 310-412 as the carboxyl terminus of the protein, also was prepared. When overexpressed transiently in COS M6 cells, the wild-type and mutant A2a receptors exhibited similar 2-[p-(2-[3H]carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine saturation binding and competition curve profiles. The following biochemical techniques confirmed that the COS M6 cells were transcribing and translating A2a receptors of the expected molecular masses: (a) immunoblotting with an antipeptide antibody directed against the putative carboxyl-terminal side of the second extracellular loop (Tyr155-Val172) of the canine A2a adenosine receptor, (b) photoaffinity labeling with the A2a-selective agonist 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine (125I-azido-PAPA-APEC), and (c) partial purification of the hexahistidine-tagged receptor on Ni2+.nitrilotriacetic acid resin. A presumed A2a receptor (44 kDa) from rabbit striatal membranes also was detected with the antisera against amino acids Tyr155-Val172 of the RDC8 receptor. Not only could the mutant A2a receptor be photolabeled specifically with 125I-azido-PAPA-APEC but so too could unglycosylated A2a receptors (i.e., from tunicamycin-treated COS M6 cells), either full length or truncated. In all of these cases, photolabeling was attenuated by both agonist and antagonist competitors.