Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity.

Microcystin-LR is a hepatotoxin produced by several cyanobacteria. Its toxicity is mainly due to a inhibition of protein phosphatase, PP1 and PP2A. Previously, we used a cell line stably expressing uptake transporter for microcystin-LR, OATP1B3 (HEK293-OATP1B3 cells). In this study, to determine whether overexpression of carboxylesterase (CES), which degrades ester-group and amide-group, attenuates the cytotoxicity of microcystin-LR, we generated the HEK293-OATP1B3/CES2 double-transfected cells. HEK293-OATP1B3/CES2 cells showed high hydrolysis activity of p-nitrophenyl acetate (PNPA), which is an authentic substrate for esterase. CES activity in HEK293-OATP1B3/CES2 cells was approximately 3-fold higher than that in the HEK293-OATP1B3 cells. HEK293-OATP1B3/CES2 cells (IC50: 25.4±7.7nM) showed approximately 2.1-fold resistance to microcystin-LR than HEK293-OATP1B3 cells (IC50: 12.0±1.5nM). Moreover, the CES inhibition assay and microcystin-agarose pull down assay showed the possibility of the interaction between CES2 and microcystin-LR. Our results indicated that the overexpression of CES2 attenuates the cytotoxicity of microcystin-LR via interaction with microcystin-LR.

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3.

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown. In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.

New acylated anthocyanins from purple yam and their antioxidant activity.

Purple yam (Dioscorea alata L.), which is widely distributed in tropical and subtropical regions, is characterized by its color and viscosity. Previous studies have shown that purple yams contain a variety of acylated anthocyanins that exhibit higher levels of antioxidant activity than the corresponding nonacylated compounds. In this study, the pigments found in purple yams from the Philippines (D. alata) were isolated and evaluated in terms of antioxidant activity. Four new acylated anthocyanins, alanins (1-4) were isolated from the MeOH extracts of purple yam, which were subsequently determined to be cyanidin (1, 2, and 4) and peonidin (3) type compounds, along with four known anthocyanins (5-8). The structures of 1-4 were determined by spectroscopic methods, including NMR and MS analyses. The antioxidant activities of anthocyanins 1-8 were investigated using oxygen radical absorbing capacity and ferric reducing antioxidant power assays.

Naringin attenuates the cytotoxicity of hepatotoxin microcystin-LR by the curious mechanisms to OATP1B1- and OATP1B3-expressing cells.

Microcystin-LR, which is an inhibitor of serine/threonine protein phosphatase (PP)1 and PP2A, induces liver injury by its selective uptake system into the hepatocyte. It is also thought that microcystin-LR induces reactive oxygen species (ROS). We tried to establish the chemical prevention of microcystin-LR poisoning. We investigated the effect of grapefruit flavanone glycoside naringin on cytotoxicity of microcystin-LR using human hepatocyte uptake transporter OATP1B3-expressing HEK293-OATP1B3 cells. We found cytotoxicity of microcystin-LR was attenuated by naringin in a dose dependent manner. The inhibition magnitude of total cellular serine/threonine protein phosphatase activity induced by microcystin-LR was suppressed by naringin. In addition, uptake of microcystin-LR into HEK293-OATP1B3 cells was inhibited by naringin. Furthermore, microcystin-LR induced phosphorylation of p53 was inhibited by naringin. Regardless of the difference in the exposure pattern of pre-processing and post-processing of naringin, the toxicity of microcystin-LR was comparable. These results suggested that naringin is promising remedy as well as preventive medicine for liver damage with microcystin-LR. In addition, involvement of ROS production after exposure to the sublethal concentrations of microcystin-LR in the onset of cytotoxicity was negligible. Therefore, inhibition of microcystin-LR uptake and the pathway other than ROS production would be involved in the effect of naringin on the attenuation of microcystin-LR toxicity.

Microcystin-LR induces anoikis resistance to the hepatocyte uptake transporter OATP1B3-expressing cell lines.

Microcystin-LR is a cyclic peptide released by several bloom-forming cyanobacteria. Understanding the mechanism of microcystin-LR toxicity is important, because of the both potencies of its acute cytotoxicity and tumor-promoting activity in hepatocytes of animals and humans. Recently, we have reported that the expression of human hepatocyte uptake transporter OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. In this study, we demonstrated a novel function of microcystin-LR which induced bipotential changes including anoikis resistance and cytoskeleton reorganization to OATP1B3-transfected HEK293 cells (HEK293-OATP1B3). After exposure to microcystin-LR, HEK293-OATP1B3 cells were divided to the floating cells and remaining adherent cells. After collection and reseeding the floating cells into a fresh flask, cells were confluently proliferated (HEK293-OATP1B3-FL) under the microcystin-LR-free condition. Both the proliferated HEK293-OATP1B3-FL and remaining adherent HEK293-OATP1B3-AD cells changed the character with down- and up-regulation of E-cadherin, respectively. Additionally, these cells acquired resistance to microcystin-LR. These results suggest that microcystin-LR could be associated with not only tumor promotion, but also epithelial-mesenchymal transition-mediated cancer metastasis. Furthermore, microcystin-LR might induce the cytoskeleton reorganization be accompanied epithelial-mesenchymal transition.

Component analysis of propolis collected on Jeju Island, Korea.

A study of propolis from Jeju Island, located off the southern tip of Korea, led to the isolation and identification of eight chalcones: (±)-(E)-4'-methoxy-4,2'-dihydroxy-3'-(2″,3″-dihydroxy-3″-methylbutyl)-chalcone, (E,E,E)-4,2',4'-trihydroxy-3'-(7″-hydroxy-3″,7″-dimethyloct-2″,5″-dienyl)-chalcone, (±)-(E,E)-4,2',4'-trihydroxy-3'-(5″-hydroxy-3″,7″-dimethyloct-2″,6″-dienyl)-chalcone, (±)-(E)-4'-methoxy-4,3″,4″-trihydroxy-2″,2″-dimethyldihydropyrano-(2',3')-chalcone, (±)-(E)-4'-methoxy-4,3″-dihydroxy-2″-(1″'-hydroxyisopropyl)-dihydrofurano-(2',3')-chalcone, (-)-(E)-4,4'-dihydroxy-2″-(1″'-hydroxy-1″',5″'-dimethylhex-4″'-enyl)-dihydrofurano-(2',3')-chalcone, (+)-(E)-4,2'-dihydroxy-2″-methyl-2″-(3″',4″'-dihydroxy-4″'-methylpentanyl)-2H-pyrano-(3',4')-chalcone and (-)-(E)-4,2'-dihydroxy-2″-methyl-2″-(3″',4″'-dihydroxy-4″'-methylpentanyl)-2H-pyrano-(3',4')-chalcone. Nineteen other known compounds were also isolated. Their structures were determined by spectroscopic analyses and comparison with literature data. The propolis from Jeju Island contained compounds not present in propolis from other regions.

Identification of the plant origin of propolis from Jeju Island, Korea, by observation of honeybee behavior and phytochemical analysis.

Propolis collected on Jeju Island, Korea, contains characteristic components not present in propolis from other regions. Hence, the plant origin of the propolis from Jeju Island can be expected to be a novel plant. To identify the plant origin of this propolis, first we observed honeybee behavior, and found them collecting the resin from Angelica keiskei. Then comparative analyses of chemical and biological properties of the resin from the plant and propolis from hives of nearby apiaries were performed. Alcoholic extracts showed entirely identical HPLC profiles and closely similar antioxidant activities. These results indicate that A. keiskei is the plant origin of the propolis from Jeju Island, Korea.

Isolation of methyl syringate as a specific aflatoxin production inhibitor from the essential oil of Betula alba and aflatoxin production inhibitory activities of its related compounds.

Methyl syringate was isolated from the essential oil of Betula alba as an aflatoxin production inhibitor. It inhibited aflatoxin production of Aspergillus parasiticus and Aspergillus flavus with IC(50) values of 0.9 and 0.8 mM, respectively, without significantly inhibiting fungal growth. Methyl syringate reduced mRNA levels of genes (aflR, pksA, and omtB) [corrected] encoding proteins required for aflatoxin biosynthesis. Methyl gallate, methyl 3,4,5-trimethoxybenzoate, and methyl 3-O-methylgallate inhibited both aflatoxin production and fungal growth of A. parasiticus and A. flavus. However, their acids and syringic acid did not inhibit aflatoxin production and growth of A. parasiticus significantly, although gallic acid inhibited aflatoxin production of A. flavus with selectivity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of methyl syringate was much weaker than that of gallic acid. These results showed that methyl syringate has a unique inhibitory activity toward aflatoxin production with a different mode of action from that of gallic acid.

Surugapyrone A from Streptomyces coelicoflavus strain USF-6280 as a new DPPH radical-scavenger.

Surugapyrone A, a novel 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging 2-pyrone, was isolated from the culture filtrate of an actinomycete strain USF-6280. This strain was assigned to the genus Streptomyces owing to its morphological and chemotaxonomic characteristics. Phylogenetic analysis based on 16S ribosomal RNA gene sequences supported the classification of the producing strain as a member of the genus Streptomyces and indicated that it was closely related to Streptomyces coelicoflavus. As the morphological, physiological and biochemical characteristics of the producing strain were in agreement with those of the type strain of S. coelicoflavus, we concluded that strain USF-6280 should be identified as a member of S. coelicoflavus. The structure of surugapyrone A was determined to be 4-hydroxy-6-isopropyl-3-methyl-2-pyrone on the basis of the spectroscopic data. The results of feeding experiments with (13)C-labeled compounds indicated that surugapyrone A was biosynthesized through a polyketide pathway involving isobutyrate, acetate and propionate.

Isolation of 2,4,4'-trihydroxydeoxybenzoin and 3'-hydroxydaidzein from soybean miso.

Two 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging compounds were isolated from soybean miso. They were determined to be 2,4,4'-trihydroxydeoxybenzoin and 3'-hydroxydaidzein on the basis of spectroscopic data. In the manufacturing process for soybean miso, 2,4,4'-trihydroxydeoxybenzoin appeared during culture aging, and the quantity of it increased in a time-dependent manner.

Soybean lipoxygenase inhibitory and DPPH radical-scavenging activities of aspernolide A and butyrolactones I and II.

Aspernolide A and butyrolactones I and II showed inhibitory activities against soybean lipoxygenase. All of them also had DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity. An analysis of the mechanism for radical scavenging allowed us to deduce that aspernolide A was converted to a quinone methide by a reaction with two molecules of the DPPH radical.

New potent DPPH radical scavengers from a marine-derived actinomycete strain USF-TC31.

Six compounds were isolated as radical scavengers from the culture broth of a marine-derived actinomycete strain USF-TC31. The structures of two novel compounds were determined to be those of N-carbamoyl-2,3-dihydroxybenzamide (5) and 2-acetamido-3-(2,3-dihydroxybenzoylthio)propanoic acid (6), and four known compounds were identified to be anthranilic acid (1), 2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3) and benadrostin (4) on the basis of spectroscopic data. Compound 6 was characterized as a racemate by its specific rotation. Each of the obtained compounds was evaluated for DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, and compounds 2, 3, 5 and 6 each exhibited potent activity in comparison with the butylhydroxytoluene (BHT) positive control.

Indole derivatives from a marine sponge-derived yeast as DPPH radical scavengers.

Two new indole derivatives (3, 4) and three known compounds (1, 2, 5) were isolated as radical scavengers from the culture filtrate of a marine sponge-derived yeast. Their structures were determined to be tyrosol (1), tryptophol (2), 2-(1H-indol-3-yl)ethyl 2-hydroxypropanoate (3), 2-(1H-indol-3-yl)ethyl 5-hydroxypentanoate (4), and cyclo(L-Pro-L-Tyr) (5) on the basis of their spectroscopic data. The absolute configurations of compounds 3 and 5 were determined by chiral HPLC analysis combined with synthesis and Marfey's method, respectively. Each obtained compound was evaluated for DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, and all compounds exhibited weak activities.

Isolation of a new metabolite from biotransformation of daidzein by Aspergillus oryzae.

Two compounds were isolated from biotransformation of daidzein, a soybean isoflavone, by Aspergillus oryzae. One was 6,7,8,4'-tetrahydroxyisoflavone, an A-ring dihydroxylated daidzein, and the other was a novel compound which had the cleaved B-ring of daidzein. The former was perhaps derived from 8-hydroxydaidzein and/or 6-hydroxydaidzein, and had high DPPH radical-scavenging activity.

Novel secondary metabolites, spirosorbicillinols a, B, and C, from a fungus.

In our searching program for novel sorbicillin related compounds, three novel compounds, spirosorbicillinols A (1), B (2), and C (3), were isolated from the fermentation broth of the USF-4860 strain isolated from a soil sample. The planar structures of compounds 1-3 were determined from spectroscopic evidence and degradation reaction, and that of 1 was the same as that of 2. The relative stereochemistries of compounds 1-3 were determined by (1)H-(1)H coupling constants, the elucidation of HMBC and NOESY spectra in detail. 1 and 2 were stereoisomers at C8 position, each other. We propose that compounds 1 and 2 were formed by exo and endo intermolecular Diels-Alder reaction between sorbicillinol as a diene and scytolide (proposed precursor-1) as a dienophile, respectively. Similarly, we propose that compound 3 was formed by an endo intermolecular Diels-Alder reaction between sorbicillinol and proposed precursor-2.

Surugapyrroles A and B, two new N-hydroxypyrroles, as DPPH radical-scavengers from Streptomyces sp. USF-6280 strain.

Two new N-hydroxypyrroles, surugapyrroles A and B, were isolated as DPPH radical-scavengers from a culture broth of Streptomyces sp. USF-6280 strain. The structures of the two compounds were elucidated by spectroscopic evidences and derivatization. Their DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity was as high as that of butylhydroxytoluene (BHT): surugapyrrole A, ED(50) 50.3 microM; surugapyrrole B, ED(50) 75.8 microM.

Biosynthesis of tetrapetalones.

The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was studied by feeding experiments with [1-13C] sodium propanoate, [1-13C] sodium butanoate, [carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and [1-13C] glucose, followed by analysis of the 13C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1) is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the biosynthetic relationship of the four tetrapetalones was indicated.

Novel DPPH radical scavengers, demethylbisorbibutenolide and trichopyrone, from a fungus.

In our screening program for antioxidants with 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging activity, two novel compounds, demethylbisorbibutenolide (1) and trichopyrone (2), were isolated from the fermentation broth of the fungus of USF-4860 strain isolated from a soil sample. The structures of these compounds were determined from spectroscopic evidence. The biosynthetic origin of the carbon atoms of 2 was unambiguously determined by feeding experiments using (13)C-labeled precursors and elucidation of the (13)C-NMR spectrum of (13)C-labeled 2. These studies showed that 2 was derived from five acetates and a methyl group of methionine. In the DPPH-radical scavenging assay, 1 and 2 gave ED(50) values of 149 and 167 muM after standing for 2.0 hr. Compound 2 reacted with the DPPH radical to form reaction product 3 which was determined to be 1-[4-(3,4-dihydro-3-methyl-6-{1,3-pentadienyl}-2,4-dioxo-2H-pyran-3-yl)-phenyl]-1-phenyl-2-picrylhydrazine from spectroscopic evidence.

Changes in the mutagenic and estrogenic activities of bisphenol A upon treatment with nitrite.

Bisphenol A (4,4'isopropylidenediphenol: BPA), an endocrine-disrupting chemical, is contained in food-packaging and can-coating agents as well as in dental sealants. Nitrite is present in vegetables, fish and tap water as an ingredient or contaminant, and also in human saliva. Here, we explored the possible generation of genotoxicity from the reactions of BPA and nitrite under acidic conditions, a situation simulating the stomach. We determined the changes in the mutagenic and estrogenic activities of BPA before and after nitrite treatment. Untreated BPA did not exhibit any mutagenicity. However, the mixture of BPA and sodium nitrite after incubation at pH 3.0 showed strong mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 either with or without a metabolic activation system (S9 mix). The clastogenic properties of nitrite-treated and untreated BPA were analyzed by a micronucleus test with male ICR mice. A single gastric intubation of nitrite-treated BPA induced a significantly higher frequency of micronucleated reticulocytes (MNRETs) in mice. The results of analysis of electron spin resonance (ESR) suggest that the expression of the mutagenic activity of nitrite-treated BPA is related to the generation of radicals in the reaction mixture. By applying 1H and 13C NMR, AB-MS and APCI/LC/MS, we identified two compounds 3-nitrobisphenol A and 3,3'-dinitro-bisphenol A. These compounds were synthesized by the reaction of BPA with nitric acid. 3,3'-Dinitro-bisphenol induced a significantly greater frequency of MNRETs in male ICR mice. By applying a green fluorescent protein (GFP)-reporter expression system and an estrogen R(alpha) competitor screening kit, we found that nitrite-treated BPA and 3,3'-dinitro-bisphenol A showed weak estrogenic activity compared to that of untreated BPA.

DPPH radical-scavenging compounds from dou-chi, a soybean fermented food.

Dou-chi, a traditional soybean food fermented with Aspergillus sp., is usually used as a seasoning in Chinese food, and has also been used as a folk medicine in China and Taiwan. As 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengers, four phenol compounds, one isoflavanone, eight isoflavones and one 4-pyrone have been isolated from dou-chi. Among these fourteen compounds, 3'-hydroxydaidzein, dihydrodaidzein and a 4-pyrone compound have not yet been isolated from soybean miso. The structure of the novel 4-pyrone compound, 3-((E)-2-carboxyethenyl)-5-(4-hydroxyphenyl)-4-pyrone-2-carboxylic acid was elucidated by using the same compound as that obtained from the biotransformation of daidzein. 3'-Hydroxydaidzein showed as high DPPH radical-scavenging activity as that of alpha-tocopherol, and 6-hydroxydaidzein had mushroom tyrosinase inhibitory activity with an IC(50) value of 10 muM. The order of estrogenic activity is as follows: genistein > daidzein >> 3'-hydroxydaidzein > 8-hydroxygenistein, using a green fluorescent protein expression system. Furthermore, the contents of isoflavones in the fermentation process of dou-chi were measured.