Purification and properties of an insecticidal metalloprotease produced by Photorhabdus luminescens strain 0805-P5G, the entomopathogenic nematode symbiont.

A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5-10, and 14-60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella) of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.

Kaempferol inhibits enterovirus 71 replication and internal ribosome entry site (IRES) activity through FUBP and HNRP proteins.

Flavonoids are associated with multiple biological and pharmacological activities, including anti-enterovirus activity. An internal ribosomal entry site (IRES) required for viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation translation initiation requires the interaction of IRES specific trans-acting host factors with viral IRES element. By evaluation of 12 flavonoids against EV71 infection, we found that (a) 7,8-dihydroxyflavone, kaempferol, quercetin, hesperetin and hesperidin exhibited more than 80% of cell survival and inhibition of EV71 infection; however, no anti-oxidative effects were noted from these flavonoids; (b) among them, only 7,8-dihydroxyflavone, kaempferol and hesperetin showed 40% of viral IRES activity; (c) kaempferol interfered with EV71 virus replication and pseudotyped virus production; and (d) FUBP1, FUBP3, HNRPD, HNRH1 and HNRPF proteins are associated with EV71 5'-UTR as shown using RNA affinity pull-down assay coupled with LC-MS/MS analysis. We firstly found that kaempferol may change the composition of these IRES associated trans-acting factors, and affect IRES function and EV71 virus replication. These studies help not only to understand the IRES function but also the mechanism by which drug induced cellular proteins are acting against EV71 infection.

The covalent binding of genistein to the non-prosthetic-heme-moiety of bovine lactoperoxidase leads to enzymatic inactivation.

OBJECTIVE: Genistein, a major soy isoflavone metabolite (SIF), inactivates oxidation activity of bovine lactoperoxidase (LPO). Modification of the heme moiety of LPO by nitrogen-containing compounds has been shown to inactivate LPO. In contrast, SIF mediated inactivation of LPO does not involve a heme modification and the mechanism of SIF inhibition is poorly understood. METHODS: After inactivation of LPO by genistein in the presence of H(2)O(2), trypsin-digested LPO peptide fragments were collected and analyzed by MALDI-TOF-MS to characterize the chemical binding of genistein(s) to LPO. RESULTS: The heme moiety of LPO was not modified by genistein. A covalent binding study showed that (3)H-genistein bound to LPO with a ratio of ∼12 to 1. After HPLC analysis and peak collection, trypsin-digested peptide fragments were analyzed by MALDI-TOF-MS. The 3H-genistein co-eluted peptide fragments (RT=24 min) were putatively identified as 199IVGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer (2 259 Da), 486TPDNIDIWIGGNAEPMVER504 with two bound genistein molecules or a genistein dimer (2 663 Da), and 161ARWLPAEYEDGLALPFGWTQR182 with three bound genistein molecules or a genistein trimer (3 060 Da). The fragment with a mass of 1 792 Da (RT=36 min) was identified as 132CDENSPYR139 with three genistein molecules or a genistein trimer. CONCLUSIONS: The results suggest that LPO was inactivated by irreversible covalent binding of genistein or genistein polymers to particular peptide fragments constituting regions of the outward domain. No genistein interaction with the prosthetic heme moiety of LPO was observed.

Soy protein with and without isoflavones fails to substantially increase postprandial antioxidant capacity.

Five methods for the assessment of antioxidant capacity [whole plasma conjugated diene formation, low-density lipoprotein oxidation susceptibility, ferric-reducing ability of plasma, oxygen radical absorbance capacity and perchloric-acid-treated oxygen radical absorbance capacity (PCA-ORAC)] were used in a randomized, double blind, crossover study to determine the acute postprandial antioxidant protection imparted by the isoflavone component of soy. On separate days, 16 subjects consumed one of three isocaloric shakes containing 25 g of protein in the form of soy, with 107 mg of total aglycone units of isoflavones, soy with trace isoflavones (<4 mg) or total milk protein. Blood was collected at baseline, 4 h, 6 h and 8 h after consumption. Antioxidant capacity, serum isoflavone levels, fat-soluble antioxidants and plasma vitamin C levels were evaluated. Repeated measures analysis of variance showed no significant differences (P=.05) within treatments over time in four of five antioxidant capacity measurements. Significant differences over time between the soy with trace isoflavones and the total milk protein group were observed using the PCA-ORAC assay. It can be concluded that, on an acute basis, a significant increase in serum antioxidant capacity is not detectable following consumption of soy protein.

Fenvalerate-induced alterations in calcium homeostasis in rat ovary.

OBJECTIVE: To observe the effects of fenvalerate on calcium homeostasis in rat ovary. METHODS: Female Sprague-Dawley rats were orally given fenvalerate at daily doses of 0.00, 1.91, 9.55, and 31.80 mg/kg for four weeks. The ovary ultrastucture was observed by electron microscopy. Serum free calcium concentration was measured by atomic absorption spectrophotometry. The activities of phosphorylase a in rat ovary were evaluated by the chromatometry. The total content of calmodulin in ovary was estimated by ELISA at each stage of estrous cycle. Radioimmunoassay (RIA) was used to evaluate the level of serum progesterone. RESULTS: Histopathologically, damages of ovarian corpus luteum cells were observed. An increase in serum free calcium concentration was observed in rats treated with 31.80 mg/kg fenvalerate. The activities of phosphorylase a enhanced in all treated groups, and fenvalerate increased the total content of calmodulin significantly in estrus period. Serum progesterone levels declined in fenvalerate exposed rats in diestrus. CONCLUSION: Fenvalerate interferes with calcium homeostasis in rat ovary. Also, the inhibitory effects of fenvalerate on serum progesterone levels may be mediated partly through calcium signals.

Effects of terephthalic acid on rat lipid metabolism.

OBJECTIVE: To study the effect of terephthalic acid (TPA) on lipid metabolism in Sprague-Dawley (SD) rats. METHODS: Five groups of SD rats that ingested 0%, 0.04%, 0.2%, 1%, and 5% TPA, respectively, were included in a 90-day subchronic feeding study. Effects of TPA on levels of serum protein, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) were observed. Urine samples were collected and analyzed for concentration of ion. RESULTS: TPA decreased the level of serum T-AOC in a dose dependent manner. The contents of serum and bladder MDA significantly decreased in 1% and 5% TPA ingestion groups. Serum CuZn superoxide dismutase (CuZnSOD) lowered in groups of 0.2%, 1%, and 5% TPA. TPA subchronic feeding had no significant influences on serum TC, LDL or HDL, but increased serum TG, TP and ALB after administration of 0.04% and/or 0.2% TPA. Concentrations of urinary Ca2+, Mg2+, Na+, and K+ were elevated in 1% and 5% TPA groups. CONCLUSION: Antioxidative potential decreased after TPA exposure. MDA increase in serum and bladder tissues was one of the most important reactions in rats which could protect themselves against TPA impairment. The decrease of serum CuZnSOD was related to the excretion of Zn2+.

Metabolism of terephthalic acid and its effects on CYP4B1 induction.

OBJECTIVE: To investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder. CONCLUSION: Lack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells.

Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50-200 µg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

Genotoxic effects on spermatozoa of carbaryl-exposed workers.

Carbaryl, one of the most important insecticides, is widely produced and used. To explore carbaryl-induced genotoxic effects of spermatozoa, particularly DNA damage and chromosome aberrations (CA), we first examined conventional semen parameters, the progression and motion parameters of the spermatozoa among 16 carbaryl-exposed workers and 30 internal and external control individuals. Sperm DNA damage represented as positive percentage of DNA fragmentation was detected by a modified terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Then numerical CA of chromosome X, Y, and 18 were investigated by multicolor fluorescence in situ hybridization (FISH). The results showed significant differences in the percentage of sperm abnormality between carbaryl-exposed group and the external control group (p = 0.008). Mean (+/-SD) percentage of spermatozoa with fragmented DNA in carbaryl-exposed group (21.04 +/- 8.88%) was significantly higher than those in the internal (13.36 +/- 12.17%) and external control groups (13.92 +/- 7.15%), respectively (p = 0.035 and p = 0.030). Using FISH, we observed the frequency of sperm sex chromosome disomy was 0.661 +/- 0.238% in the exposed group, which was significantly higher than that in the external control group (0.386 +/- 0.140%) (p = 0.001), and the carbaryl-exposed group (0.276 +/- 0.126%) had an elevated chromosome 18 disomy compared with the internal (0.195 +/- 0.094%) and external control groups (0.124 +/- 0.068%), respectively (p < 0.05 and p < 0.01). In addition, carbaryl-exposed donors had significantly higher sperm nullisomic frequencies of sex chromosomes and chromosome 18 than the external controls (p < 0.01) but not the internal controls. In summary, the frequencies of aneuploidy and numerical CA showed significant differences between exposed group and control groups (p < 0.05 and/or p < 0.01). Moreover, positive correlations were found between sex chromosome disomy, aneuploidy rate, and morphologic abnormalities in spermatozoa of all donors (r = 0.564 and r = 0.555, p < 0.01). Our findings suggested that carbaryl might induce morphologic abnormalities and genotoxic defects of spermatozoa among exposed workers by causing DNA fragmentation and numerical CA in spermatogenesis as a potential genotoxicant. The evidence also indicated that the spermatotoxicity induced by carbaryl exposure might be related to adverse reproductive outcomes.

Effects of fenvalerate on progesterone production in cultured rat granulosa cells.

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate on progesterone production. Various concentrations (0, 1, 5, 25, 125 and 625 microM) of fenvalerate were added to the cell cultures for 24 h. rGCs were stimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesterone production and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results showed that fenvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presence of FSH and was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenvalerate implicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein expression of P450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependent protein kinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.

Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis.

Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 +/- 0.6 nmol/g total genistein; 3.0 +/- 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.

JWA--a novel environmental-responsive gene, involved in estrogen receptor-associated signal pathway in MCF-7 and MDA-MB-231 breast carcinoma cells.

The pyrethroid insecticide fenvalerate and the organophosphorus insecticide phoxim are now the most widely used agents for indoor pest control in China. Fenvalerate was shown to mimic estrogenic activity, whereas phoxim did not induce similar effects. Our previous studies demonstrated that JWA, a novel retinoic acid-inducible and cytoskeleton-associated gene, is also a potential environmental-responsive gene with increased expression to oxidative and heat-shock stresses. In the present study, the influence of both fenvalerate and phoxim was examined on the expression of JWA in MCF-7 (ER+) and MDA-MB-231 (ER-) human breast carcinoma cell lines. Concentrations of 0.01, 1, and 100 micromol/L of fenvalerate or phoxim were selected to treat both MCF-7 and MDA-MB-231 cells at 1, 3, and 5 d, respectively. The MTT results only showed that fenvalerate stimulated MCF-7 cell proliferation. Western blot assay was employed to detect the expressions of JWA and heat-shock proteins (hsp27 and hsp70). The results showed that after treatment with fenvalerate, both JWA and hsp70 showed similar expression patterns in the both cell lines; however, all the expression patterns of JWA, hsp27, and hsp70 were evidently reversed between ER+ and ER- cells. In addition, phoxim-treated cells showed a concentration-dependent relationship in JWA expression at all time points. These results suggest that JWA has similar functions with respect to hsp27 and hsp70, and might be a novel signal molecule in estrogen receptor-related signal transduction pathways in mammalian cells.

The roles of metallothionein on cadmium-induced testes damages in Sprague-Dawley rats.

The present study was to investigate whether metallothionein (MT) was involved in sensitivity of testis to cadmium (Cd) and protection of rats from Cd-induced testis damages. The rats were treated by intraperitoneal injection with 0.2, 0.4, and 0.8mg Cd/kg BW for 7 days. The atomic absorption spectrophotometry and cadmium hemoglobin affinity assay were applied to evaluate the contents of Cd and MT in testis and liver. The testis glutathione (GSH), malondialdehyde (MDA) and daily sperm production were measured. There were substantial increases of both Cd and MT in the liver after Cd exposure. The testis Cd and MT contents were lower than those in the corresponding liver in Cd-exposed rats. Low doses of Cd (0.2 and 0.4mg/kg BW) induced MT in testis, while a significant decline of MT was found in rats treated with 0.8mg Cd/kg BW. By a concomitant decrease of MT, there was an obvious increase of MDA and marked decreases of GSH, daily sperm production in rats treated with 0.8mg Cd/kg BW. These findings suggested MT was more difficult to be induced in the testis than in the liver by Cd, which might account for the high susceptibility of testis to Cd. MT, increased by a low dose of Cd, played an important role in protecting testis against Cd toxicity by sequestering and antioxidating.

Terephthalic acid occupational exposure and its effect on organ functions in fiber workers.

OBJECTIVES: : To investigate the exposure to terephthalic acid (TPA), and to evaluate it's effects on organ function including the potential risk factors for uroliths and bladder tumor to TPA. METHODS: : Exposure-response modeling was carried out in a cohort of 141 TPA exposure workers and three subgroups were classified according to their urine TPA concentration. The control group consisted of 77 workers with no exposure to TPA dust. The inhalatory exposure of the application workers was estimated from biological monitoring data. Urine and blood samples were collected from all workers before and after work shift to monitor variables of liver, kidney, and lung. Haematological variables and serum biochemistry were valued, pulmonary functions were tested, and ion changes in both serum and urine were measured. RESULTS: : Increased urinary excretion of TPA (0-5mmol/molCr after the shift) reflected occupational workers TPA exposure. We also observed the exposure-response relations for the intensity of TPA exposure and the urine variables. Increased serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) but within normal range is not increased. The slightly increased serum angiotensin-converting enzyme activity (SACE) was considered to be related to particulate of airborne TPA dust inhalation. No difference between referents and workers exposed to TPA was found for haematological variables. CONCLUSIONS: : No clinical organ dysfunctions were found in this investigation working with TPA. However, special precautions are still necessarily taken to avoid excessive or prolonged contact.

[Effects of mono(2-ethylhexyl) phthalate on testosterone biosynthesis in leydig cells cultured from the rat testis].

OBJECTIVE: To investigate the effects of mono(2-ethylhexyl) phthalate(MEHP), the primary metabolite of di(2-ethylhexyl) phthalate (DEHP), on testosterone biosynthesis in Leydig cells cultured from the Sprague Dawley rat testis. METHODS: Based on the primary Leydig cell culture model, MEHP exposure groups involved control (0 micromol/L), 62.5, 125, 250, 500 and 1000 micromol/L. We observed mitochondria activity with the MTT method, measured the testosterone level with RIA and determined steroidogenesis acute regulatory protein (StAR) mRNA expression with RT-PCR. RESULTS: After Leydig cells were exposed to MEHP for 24 hours, the activity of mitochondria enhanced evidently at 250 micromol/L and then declined markedly at 1000 micromol/L compared with the control group (P < 0.01). The testosterone level showed an increasing tendency in both basal and hCG-stimulated states with statistical significance at 250 and 500 micromol/L compared with the control group (P < 0.01). However, the expression of StAR mRNA appeared unchanged at 62.5, 125 or 250 micromol/L, but exhibited a decreasing tendency at 500 and 1000 micromol/L (P < 0.01). CONCLUSION: ME- HP directly affected the activity of mitochondria and testosterone biosynthesis of the Leydig cells in vitro. StAR, the regulator of cholesterol transport into mitochondria, might not be responsible for the increase of testosterone biosynthesis induced by MEHP.

Alterations of FSH-stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate.

Fenvalerate, a synthetic pyrethroid, is widely used in agriculture and other domestic applications in China. Recently, Fenvalerate has been suspected to be one of the endocrine-disrupting chemicals (EDC). In this study, we investigated the effects of fenvalerate on follicle-stimulating hormone (FSH)-stimulated progesterone (P4) production by human ovarian luteinizing-granulosa cells (hGLCs). After 24 h incubation, fenvalerate inhibited FSH-stimulated P4 production. At the same time, FSH-stimulated cAMP also decreased. Due to calcium and Ca2+ -calmodulin (CaM) system involving gonadotropin-stimulated steroidogenesis by granulosa cells, we then evaluated the effects of fenvalerate on trifluoperazine (TFP)- and verapamil-driven FSH-stimulated P4 production. The results showed that calcium or calmodulin might play a role in fenvalerate-induced alterations in FSH-stimulated P4 biosynthesis. Then, the effects of fenvalerate on calcium homeostasis in hGLCs were studied. The result showed that 5 microM fenvalerate induced a slow increase in [Ca2+]i in hGLCs by using a fluorescent Ca2+ indicator fluo-3/AM. The changes in total concentration of CaM in hGLCs induced by fenvalerate were evaluated by a method of immunofluorescence. There is a significant increase in all treated groups. In summary, fenvalerate could inhibit FSH-stimulated P4 production. Also, fenvalerate interferes with calcium homeostasis in hGLCs. The effects of fenvalerate on FSH-stimulated ovarian steroidogenesis may be mediated partly through calcium signal.

Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo.

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g. cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g. enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper reviews the evidence in humans and animals for anti-thyroid effects of soy and its principal isoflavones, genistein and daidzein.

Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats.

Isomers of 4-nonylphenol (NP), which are important industrial compounds and environmental breakdown products from widely used surfactants, have estrogenic activity in vitro and in vivo that has prompted interest in its potential for modulation of endocrine function in humans and wildlife. Mass spectrometry was used to quantify NP and metabolites in serum and endocrine-responsive tissues from dietary exposure in Sprague-Dawley rats. Tissue accumulation of NP aglycone was observed despite the predominance of glucuronidation in blood. Serum toxicokinetics of total NP, measured following gavage administration, showed rapid absorption and elimination (average half-times 0.8 and 3.5 h, respectively). NP was similarly administered by gavage to pregnant dams and total and aglycone NP were measured in dam serum and fetuses to show placental transfer into serum and brain. These data provide a basis for future correlations of biologic effects observed following dietary exposure in rats with those predicted from environmental exposures to humans.

Determination of St. John's wort components in dietary supplements and functional foods by liquid chromatography.

St. John's wort (Hypericum perforatum L.) preparations, a top-selling botanical dietary supplement used primarily as an antidepressant, has recently been used as an ingredient in some food products sold as functional foods. A rapid extraction technique followed by a liquid chromatographic (LC) method was developed to determine 4 characteristic bioactive compounds (pseudohypericin, hypericin, hyperforin, and adhyperforin) from St. John's wort in dietary supplements and functional foods to which it was added. Solid samples, including dried leaf/flower mixture, dietary supplement capsules, tea bags, puff and snack bar, were extracted with methanol by sonication. Noncarbonated, fruit-flavored drinks were centrifuged and mixed with methanol. Compounds were then determined by isocratic, reversed-phase LC with UV detection at 2 wavelengths and further identified or confirmed by photodiode array spectra and LC/mass spectrometry. Within-laboratory method variations (% RSD) were satisfactory. Very low amounts, if any, of the 4 components were found in drink and puff samples, and none was found in the snack bar. The methods developed provide a useful means for the determination of St. John's wort components in dietary supplements and functional foods.

Antrodia salmonea inhibits TNF-α-induced angiogenesis and atherogenesis in human endothelial cells through the down-regulation of NF-κB and up-regulation of Nrf2 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia salmonea (AS) is known as a traditional Chinese medicine, but very few biological activities have been reported. MATERIALS AND METHODS: The present study was aimed to investigate the anti-angiogenic and anti-atherosclerotic potential of the fermented culture broth of AS against tumor necrosis factor-α (TNF-α)-stimulated human endothelial (EA.hy 926) cells. RESULTS: The non-cytotoxic concentrations of AS significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation in EA.hy 926 cells. Furthermore, AS suppressed TNF-α-induced activity and expression of matrix metalloproteinase-9 (MMP-9), and cell-surface expression of intercellular adhesion molecule-1 (ICAM-1), which was associated with abridged adhesion of U937 leukocytes to endothelial cells. Moreover, AS significantly down-regulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor κB (NF-κB) followed by suppression of I-κB degradation and phosphorylation of I-κB kinase-α (IKKα). Notably, the protective effect of AS was directly correlated with the increased expression of hemeoxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCLC), which was reasoned by nuclear translocation and transactivation of NF-E2 related factor-2 (Nrf2)/antioxidant response element (ARE). Furthermore, HO-1 knockdown by HO-1-specific shRNA diminished the protective effects of AS on TNF-α-stimulated invasion, tube formation, and U937 adhesion in EA.hy 926 cells. CONCLUSIONS: Taken together, these results suggest that Antrodia salmonea may be useful for the prevention of angiogenesis and atherosclerosis.