Antimutagenic, antitumor and estrogen receptor binding activity of the rare plant Shortia galacifolia: An ethnobotanical and chemosystematic approach.

OBJECTIVE: Shortia and other members of the Diapensiaceae family have ethnomedicinal history in both Eastern and Western hemispheres. Based on ethnopharmacological and chemosystematic evidence, pharmacological and toxicological bioassays were conducted on the rare plant Oconee Bell, Shortia galacifolia. MATERIALS AND METHODS: Extracts were examined in assays for antimutagenicity, antitumor and estrogen receptor (ER)-binding activity. Antitumor activity was assessed by the tumor induction assay (TiA), using Agrobacterium tumefaciens based on its ability to transform plant tissue. Antimutagenicity was examined using the Ames bacterial reverse mutation test. Recombinant human ERα and ERß proteins were utilized to screen extracts for receptor selectivity. RESULTS: All concentrations of extracts inhibited A. tumefaciens-induced tumor formation on potato discs, with the mature rhizome extracts having the most marked inhibition. All three plant extracts significantly inhibited the formation of histidine-independent revertant colonies after exposure to the mutagen 2-aminoanthracene (2-AA) in the Ames Salmonella mutagenicity assay. In the ER binding assays, ERß, but not ERα, displayed affinity for Shortia extracts. CONCLUSION: Antitumor, ER binding and antimutagenic activities of S. galacifolia extracts were identified using rapid bench-top assays and warrant further investigations.

Effects of Panax ginseng, zearalenol, and estradiol on sperm function.

BACKGROUND: Estrogen signaling pathways are modulated by exogenous factors. Panax ginseng exerts multiple activities in biological systems and is classified as an adaptogen. Zearalenol is a potent mycoestrogen that may be present in herbs and crops arising from contamination or endophytic association. The goal of this study was to investigate the impact of P. ginseng, zearalenol and estradiol in tests on spermatozoal function. METHODS: The affinity of these compounds for estrogen receptor (ER)-alpha and beta (ERα and ERß)-was assessed in receptor binding assays. Functional tests on boar spermatozoa motility, movement and kinematic parameters were conducted using a computer-assisted sperm analyzer. Tests for capacitation, acrosome reaction (AR), and chromatin decondensation in spermatozoa were performed using microscopic analysis. RESULTS: Zearalenol-but not estradiol (E2)- or ginseng-treated spermatozoa-decreased the percentage of overall, progressive, and rapid motile cells. Zearalenol also decreased spontaneous AR and increased chromatin decondensation. Ginseng decreased chromatin decondensation in response to calcium ionophore and decreased AR in response to progesterone (P4) and ionophore. CONCLUSION: Zearalenol has adverse effects on sperm motility and function by targeting multiple signaling cascades, including P4, E2, and calcium pathways. Ginseng protects against chromatin damage and thus may be beneficial to reproductive fitness.

Multiple Integrated Complementary Healing Approaches: Energetics & Light for bone.

A synergistic-healing strategy that combines molecular targeting within a system-wide perspective is presented as the Multiple Integrated Complementary Healing Approaches: Energetics And Light (MICHAEL). The basis of the MICHAEL approach is the realization that environmental, nutritional and electromagnetic factors form a regulatory framework involved in bone and nerve healing. The interactions of light, energy, and nutrition with neural, hormonal and cellular pathways will be presented. Energetic therapies including electrical, low-intensity pulsed ultrasound and light based treatments affect growth, differentiation and proliferation of bone and nerve and can be utilized for their healing benefits. However, the benefits of these therapies can be impaired by the absence of nutritional, hormonal and organismal factors. For example, lack of sleep, disrupted circadian rhythms and vitamin-D deficiency can impair healing. Molecular targets, such as the Wnt pathway, protein kinase B and glucocorticoid signaling systems can be modulated by nutritional components, including quercetin, curcumin and Mg(2+) to enhance the healing process. The importance of water and water-regulation will be presented as an integral component. The effects of exercise and acupuncture on bone healing will also be discussed within the context of the MICHAEL approach.

Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa.

Multiple inter-connected signaling pathways, involving kinases and phosphatases, form a framework that controls sperm motility, function, and fertilizing ability. Methods that give a broad view of the proteomic landscape may prove valuable in uncovering new crosstalk connections, as well as in discovering new proteins within this regulatory framework. A multi-immunoblotting strategy was utilized to evaluate this concept on human and porcine spermatozoa samples. In human and porcine spermatozoa, a diversity of kinases were identified including protein kinase A (PKA), protein kinase B (PKB), isoforms of protein kinase C (PKC), calmodulin-dependent kinases (CAMK), casein kinase (CK), and isoforms of glycogen synthase kinase (GSK3). Several phosphatases, such as protein phosphatase (PP)-1, PP2A, PP2C, and mitogen activated protein kinase (MAPK) phosphatase (MKP-1), were identified in human spermatozoa. The phosphorylation epitopes recognized belonged to members of the MAPK family, in addition to α and ß isoforms of GSK3 and cAMP response element binding protein (CREB). Proteomic approaches that allow a broad view may aid in understanding the crosstalk between signaling systems in spermatozoal physiology.

Impact of kudzu and puerarin on sperm function.

The goal of this study was to investigate the impact of kudzu (Pueraria mirifica) and the isoflavone puerarin in functional toxicological tests on spermatozoa and to assess the affinity of extracts and pure isoflavones for estrogen receptor (ER)-alpha and -beta (ERα, ERß) in receptor binding assays. Capacitation, acrosome reaction and chromatin decondensation in spermatozoa were analyzed using microscopic analysis. Kudzu, but not puerarin, reduced motility of sperm. Puerarin reduced the percent spontaneous acrosome reaction in spermatozoa. The pathways used by kudzu that affect sperm function are not fully mirrored by puerarin. Puerarin, kudzu and its other phytoestrogenic components displayed preferential affinity for ERß, however the diverse effects of kudzu and puerarin on sperm function implicate the involvement of multiple signaling systems.

Mycotoxins in root extracts of American and Asian ginseng bind estrogen receptors alpha and beta.

The estrogenic activity of ginseng has been the subject of conflicting reports. Cell proliferation, induction of estrogen-responsive genes, and isolated cases of adverse reactions such as postmenopausal vaginal bleeding and gynecomastia have been reported after ginseng treatment. Other studies report antiproliferative effects with no induction of estrogen-responsive genes. We developed estrogen receptor (ER) alpha and ER alpha competitive binding assays using recombinant receptors and [(3)H]-17 alpha-estradiol to detect phytoestrogens in extracts of Asian ginseng root (Panax ginseng C. A. Meyer) and American ginseng root (Panax quinquefolius L.). Root extracts contained substances that bound both receptor isoforms. These substances had a two to three times greater affinity for ER alpha. Significantly higher binding was found in methanol extracts than in hot water extracts. Subsequent analysis of the extracts revealed significant ER binding attributable to zearalenone, the estrogenic mycotoxin produced by several Fusarium species. The ER showed no binding affinity for Rb1 and Rg1, the major ginsenosides found in P. quinquefolius and P. ginseng, respectively. Thus, ginseng extraction methods, plant species tested, and mycotoxin contaminants may help to explain the disparate literature reports. The prevalence and health significance of fungal contamination in herbal products used for medicinal purposes should be further investigated.

The contribution of hepatic steroid metabolism to serum estradiol and estriol concentrations in nonylphenol treated MMTVneu mice and its potential effects on breast cancer incidence and latency.

The two major pathways for the metabolism of estradiol-17beta (E2) are the 2- and 16-hydroxylase pathways. Research has suggested that the increased production of the estrogenically active 16-hydroxy products such as estriol (E3) may be involved in increased susceptibility to breast cancer. 4-Nonylphenol (4-NP) is an environmental estrogen that also can activate the pregnane-X receptor (PXR) and induce P-450 enzymes responsible for the production of E3. It is hypothesized that 4-NP may act in part as an environmental estrogen by increasing E3 production. Based on its affinity for the estrogen receptor (ER) alone, 4-NP may be more potent than predicted at increasing mammary cancer incidence in the MMTVneu mouse. Female mice were treated per os for 7 days at 0, 25, 50 or 75 mg kg(-1) day(-1) 4-NP to investigate the effects of 4-NP on hepatic estrogen metabolism after an acute treatment. 4-Nonylphenol increased the hepatic formation of E3 in a dose-dependent manner. However, serum E3 concentrations were only increased at 25 mg kg(-1) day(-1) presumably due to direct inhibition of E3 formation by 4-NP. MMTVneu mice were then treated for 32 weeks at 0, 30 or 45 mg kg(-1) day(-1) 4-NP to determine its effects on mammary cancer formation and estrogen metabolism. 4-Nonylphenol increased mammary cancer formation in the MMTVneu mice at 45 mg kg(-1) day(-1) but not at 30 mg kg(-1) day(-1). Mice treated with an equipotent dose of E2, 10 microg kg(-1) day(-1), based on the relative binding affinities of nonylphenol and estradiol for ER alpha, did not develop mammary cancer. This suggests that nonylphenol is more potent than predicted based on its affinity for the estrogen receptor. However, no changes in serum E3 concentrations or hepatic E3 production were measured after the chronic treatment. Changes in E3 formation were correlated with increased CYP2B levels after the 7 day 4-NP treatment, and repression of CYP2B and CYP3A after 32 weeks of 4-NP treatment. Microarray analysis and Q-PCR of liver mRNA from the mice treated for 32 weeks demonstrated a decrease in RXR alpha, the heterodimeric partner of the PXR, which may in part explain the repressed transcription of the P450s measured. In conclusion, 4-NP treatment for 32 weeks increased mammary cancer formation at a dose of 45 mg kg(-1) day(-1). However, chronic treatment with 4-NP did not increase hepatic E3 formation or serum E3 concentrations. The transient induction by 4-NP of hepatic E3 formation and serum concentrations is most likely not involved in the increased incidence of mammary cancer in MMTVneu mice since E3 serum concentrations were only increased at 25 mg kg(-1) day(-1), a dose that was not sufficient to induce mammary tumor formation. Nevertheless, the induced hepatic E3 production in the acute exposures to 4-NP was indicative of an increase in mammary cancer incidence after the chronic exposure.