In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana.

Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium-mediated transient expression. RNA-sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA-sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone-induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

AMPK activation by pterostilbene contributes to suppression of hepatic gluconeogenic gene expression and glucose production in H4IIE cells.

Pterostilbene, a bioactive component of blueberries and grapes, shows structural similarity to resveratrol, and exhibits antioxidant, anti-inflammatory, anti-cancer, hypoglycemic, and cholesterol lowering effects. Recent evidence indicates that pterostilbene is an agonist of the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPAR-α). Since PPAR-α agonists induce peroxisomal proliferation and fatty acid oxidation, we examined gene expression of acyl CoA oxidase (ACO) and carnitine palmitoyl transferase-1 (CPT-1). Pterostilbene treatment, at concentrations that demonstrated over 75% cell viability (20 µM, 50 µM), significantly increased gene expression of ACO, CPT-1, and PPAR-α. Pterostilbene treatment (50 µM) also demonstrated potent activation of AMP-activated kinase (AMPK), compared to AICAR (0.5 mM) or metformin (2 mM), consistent with upregulation in fatty acid oxidation gene expression. Since AMPK activators mimic the actions of insulin by repressing hepatic gluconeogenesis, we examined pterostilbene's effects on hepatic gluconeogenic gene expression. Pterostilbene treatment significantly repressed dexamethasone-induced phosphoenol pyruvate carboxykinase (PEPCK) and glucose6-phosphatase (G6Pase) gene expression, and decreased glucose production in H4IIE cells. Taken together, our studies demonstrate that pterostilbene, a natural compound and PPAR-α agonist, modulate several AMPK-dependent metabolic functions. The results of the present study suggest that pterostilbene may have beneficial effects in the prevention and management of type 2 diabetes and related disorders. In this study, we found that pterostilbene activated AMP-activated kinase (AMPK) and increased the expression of fatty acid oxidation genes, including acyl CoA oxidase and carnitine palmitoyl transferase-1.

A cytochrome P450 CYP71 enzyme expressed in Sorghum bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone.

Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring. A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450-like sequences, which were then analyzed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to identify those preferentially expressed in root hairs. Full-length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)-mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta. A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5-pentadecatrienyl resorcinol-3-methyl ether as substrate, as determined by gas chromatography-mass spectroscopy (GC-MS). RNAi-mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events. Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.

A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway.

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases.

Synthesis and Biological Evaluation of 3,5-Dimethoxystilbene Analogs.

In our continuing effort to discover natural product-based pest management agents, derivatives of 3,5-dimethoxystilbene were synthesized yielding 27 new and six known compounds. Compounds 11 and 12 showed strong Aedes aegypti larvicidal activity (LC50 45.31 and 49.93 µm, respectively). Furthermore, 11 and 12 exhibited high effectiveness against larvae of pesticide-susceptible and pyrethroid-resistant strains of Ae. aegypti; activity against the adult mosquitoes was low. Compounds 6f, 6g, and 6i at either 83.3 or 166.7 µg/ml reduced the mobility of second-stage juveniles (J2) of the root-knot nematode (Meloidogyne incognita) that hatched from eggs immersed in the test compounds for 7 days. However, there was little or no effect on J2 placed directly into these compounds, and none of the analogs suppressed M. incognita egg hatch. The compounds were tested for inhibition of some agriculturally important fungi; 6a, 7a, and 7e demonstrated strong inhibition of Colletotrichum species. Activity of the stilbenes against some human pathogens was also explored; 11, 12, and 16 showed moderate inhibitory activity against Cryptococcus neoformans, Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare. Except for 11 and 12, which were active against mosquito larvae and some human pathogens, no single analog demonstrated activity in all the tests, indicating specific activities. Synthesis of the analogs and structure-activity relationships are discussed.

Evaluation of PPARα activation by known blueberry constituents.

BACKGROUND: Anthocyanins are known to have hypolipidemic properties. It was deemed necessary to determine whether major blueberry anthocyanins and catechins are ligands for the transcription factor peroxisome proliferator activated receptor alpha isoform (PPARα), and compare activation with known PPARα agonistic constituents, pterostilbene and resveratrol. It was also considered important to investigate the effect of pterostilbene on PPARα gene expression, and relate results with hepatic mRNA PPARα expression up-regulation observed previously in hamsters fed a diet supplemented with blueberry peels extract (BBX). RESULTS: The anthocyanins and catechins did not activate PPARα. Only pterostilbene exhibited a dose-dependent activation of PPARα in H4IIEC3 cells. The resveratrol responses were lower than those of pterostilbene. Pterostilbene significantly and dose-dependently (at 10, 20 and 50 µmol L(-1) ) increased PPARα gene expression and the effect at 10 µmol L(-1) was greater than 100 and 200 µmol L(-1) of fenofibrate. Analysis of BBX showed levels of pterostilbene and resveratrol at 418 and 2381 ng g(-1), respectively. CONCLUSION: Anthocyanins and catechins do not appear to contribute to the up-regulation of hepatic PPARα expression observed in hamsters. While pterostilbene and resveratrol demonstrated PPARα activation, their levels in BBX do not seem to be at efficacious concentrations. These stilbenes may contribute to the up-regulation of PPARα expression by acting synergistically with each other or with other constituents in BBX.

Activities of wogonin analogs and other flavones against Flavobacterium columnare.

In our on-going pursuit to discover natural products and natural product-based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM-00-173 and BioMed) using a rapid bioassay. The flavonoids chrysin (1a), 5,7-dihydroxy-4'-methoxyflavone (11), isorhamnetin (26), luteolin (27), and biochanin A (29), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM-00-173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4'-dihydroxy-7-methoxyflavone), 26, and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24-h 50% inhibition concentration (IC50 ) results revealed that 27 and 29 were the most active compounds against F. columnare ALM-00-173 (IC50 of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC50 of 9.2 and 3.5 mg/l, resp.). These IC50 results were lower than those obtained for wogonin against F. columnare ALM-00-173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest.

Trimethoxy-resveratrol and piceatannol administered orally suppress and inhibit tumor formation and growth in prostate cancer xenografts.

BACKGROUND: Resveratrol (Res) is recognized as a promising cancer chemoprevention dietary polyphenol with antioxidative, anti-inflammatory, and anticancer properties. However, the role of its analogues in prostate cancer (PCa) chemoprevention is unknown. METHODS: We synthesized several natural and synthetic analogues of Res and characterized their effects on PCa cells in vitro using a cell proliferation assay. A colony formation assay and in vitro validation of luciferase (Luc) activity was done for LNCaP-Luc cells that were consequently used for in vivo studies. The efficacy of Res, trimethoxy-resveratrol (3M-Res) and piceatannol (PIC) was studied in a subcutaneous (s.c.) model of PCa using oral gavage. Tumor progression was monitored by traditional caliper and bioluminescent imaging. The levels of cytokines in serum were examined by ELISA, and the levels of compounds in serum and tumor tissues were determined by gas chromatography-mass spectrometry. RESULTS: We examined the anti-proliferative activities of Res/analogues in three PCa cell lines. We further compared the chemopreventive effects of oral Res, 3M-Res, and PIC in LNCaP-Luc-xenografts. We found that 2 weeks pretreatment with the compounds diminished cell colonization, reduced tumor volume, and decreased tumor growth in the xenografts. Both 3M-Res and PIC demonstrated higher potency in inhibiting tumor progression compared to Res. Notably, 3M-Res was the most active in inhibiting cell proliferation and suppressing colony formation, and its accumulation in both serum and tumor tissues was the highest. CONCLUSIONS: Our findings offer strong pre-clinical evidence for the utilization of dietary stilbenes, particularly 3M-Res, as novel, potent, effective chemopreventive agents in PCa.

Alkylresorcinol synthases expressed in Sorghum bicolor root hairs play an essential role in the biosynthesis of the allelopathic benzoquinone sorgoleone.

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3Delta(9,12,15) fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Anxiolytic action of pterostilbene: involvement of hippocampal ERK phosphorylation.

Pterostilbene, a natural analog of resveratrol, has diverse health-beneficial properties. However, the neurological activities of this compound are largely unexplored. Here, we report that pterostilbene shows anxiolytic-like actions by down-regulating phosphorylated levels of extracellular regulated kinases in the hippocampus of mice. Adult male mice administered pterostilbene (1-10 mg/kg, p. o.) were subjected to the elevated plus maze test. Pterostilbene manifested anxiolytic activity at 1 and 2 mg/kg doses, demonstrated by increases in % permanence time and number of open arm entries. The locomotor activity of the animals was unaffected at all doses. Western blot analysis revealed a decrease in both extracellular regulated kinase 1 and extracellular regulated kinase 2 phosphorylation in hippocampal homogenates from mice treated with 1 and 2 mg/kg pterostilbene. Moreover, pterostilbene was detected in the plasma and brains of mice following single oral administration. Anxiolytic activity was not observed at the higher doses (5 and 10 mg/kg). However, no impairment of motor function was observed either, suggesting a favorable safety index for the compound. These results suggest that pterostilbene has the potential for therapeutic drug development for anxiety disorders.

Scutellaria extract and wogonin inhibit tumor-mediated induction of T(reg) cells via inhibition of TGF-ß1 activity.

A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-ß1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-ß1 and T(reg) cell frequency as well as peripheral blood TGF-ß1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-ß1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3ß and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-ß1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-ß1 secretion as well as via inhibition of T cells' response to TGF-ß1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression.

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

Design, synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1.

A series of trans-stilbene derivatives containing 4'-methylthio substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. Additionally, for CYP1A2 and CYP1B1 molecular docking analysis was carried out to provide information on enzyme-ligand interactions and putative site of metabolism. 3,4,5-Trimethoxy-4'-methylthio-trans-stilbene, an analogue of DMU-212 (3,4,5,4'-tetramethoxy-trans-stilbene) was an effective inhibitor of all CYP1 enzymes. On the other hand, 2,3,4-trimethoxy-4'-methylthio-trans-stilbene, appeared to be the most selective inhibitor of the isozymes CYP1A1 and CYP1B1, displaying extremely low affinity towards CYP1A2. Molecular modeling suggested that the most probable binding poses of the methylthiostilbene derivatives in CYP1A2 active sites are those with the methylthio substituent directed towards the heme iron. Products of CYP1A2-catalyzed oxidation of 2,4,5-trimethoxy-4'-methylthiostilbene and 3,4,5-trimethoxy-4'-methylthiostilbene were identified as monohydroxylated compounds. Other studied derivatives appeared to be poor substrates of CYP1A2. Structure-activity relationship analysis rendered better understanding of the mechanism of action of xenobiotic-metabolizing enzymes crucial at the early stage of carcinogenesis.

Dietary intake of pterostilbene, a constituent of blueberries, inhibits the beta-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats.

Stilbenes are phytochemicals present in grapes, berries, peanuts and red wine. A widely studied stilbene, resveratrol (trans-3,5,4'-trihydroxystilbene), has been shown to exert antioxidant, anti-inflammatory, chemopreventive and antiaging effects in a number of biological systems. We reported earlier that pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), a structurally related stilbene found in blueberries, was effective in reducing the incidence and multiplicity of aberrant crypt foci formation in the colon of rats injected with azoxymethane (AOM). Our present study was to identify the chemopreventive potential of pterostilbene with colonic tumor formation as an end point and further to evaluate the mechanistic action of pterostilbene during colon carcinogenesis. F344 rats were given two AOM injections subcutaneously when they were 7 and 8 weeks old and continuously fed the control or 40 p.p.m. pterostilbene diet for 45 weeks. Overall analyses indicated that pterostilbene reduced colon tumor multiplicity of non-invasive adenocarcinomas, lowered proliferating cell nuclear antigen and downregulated the expression of beta-catenin and cyclin D1. Pterostilbene decreased mucosal levels of the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-4. Colon tumors from pterostilbene-fed animals showed reduced expression of inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the nuclear factor-kappaB pathway. In HT-29 cells, pterostilbene reduced the protein levels of beta-catenin, cyclin D1 and c-MYC, altered the cellular localization of beta-catenin and inhibited the phosphorylation of p65. Our data with pterostilbene in suppressing colon tumorigenesis, cell proliferation as well as key inflammatory markers in vivo and in vitro suggest the potential use of pterostilbene for colon cancer prevention.

Synthesis and biological evaluation of retinoid-chalcones as inhibitors of colon cancer cell growth.

Based on the observed anticancer activity of chalcones and retinoids, a novel class of retinoid-chalcone hybrids was designed and synthesized. As part of our ongoing studies to discover natural product based anticancer compounds, the retinoid-chalcone hybrids were tested against the colon cancer cell line HT-29. Retinoid like moiety was introduced through Friedel-Crafts alkylation of toluene. Among the synthesized compounds, the cyano derivative (E)-3-(3-oxo-3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-enyl)benzonitrile 8 showed submicromolar inhibitory activity with an IC(50) of 0.66 µM.

Antioxidant effect of trans-resveratrol, pterostilbene, quercetin and their combinations in human erythrocytes in vitro.

There is evidence that a diet rich in fruit and vegetables may reduce the risk of cancer and other degenerative diseases. However, potential health impact of bioactive phytochemicals is limited by their low amount and relatively poor bioavailability. It has been suggested that the health benefits associated with fruit and red wine consumption could be due to the whole antioxidant pool of the diet microcomponents. In this study, the antioxidant activities of trans-resveratrol, pterostilbene and quercetin, and the effect of their combination were investigated in human erythrocytes in vitro. H(2)O(2)-induced lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive species. Quercetin and pterostilbene protected erythrocyte membranes against lipid peroxidation (IC(50) values = 64 +/- 8.7 microM and 44.5 +/- 7.8 microM, respectively). Resveratrol was significantly less effective. However, the three compounds protected the erythocytes against hemolysis and GSH (reduced glutathione) depletion to the same extent. Combinations consisting of two compounds (molar ratio 1:1) influenced lipid peroxidation in a concentration-dependent manner. At lower concentrations, resveratrol with quercetin or pterostilbene inhibited synergistically the oxidative injury of membrane lipids At higher concentrations, an additive effect was observed. These protective effects may partially explain the health benefit of these bioactive microcomponents when together in the diet.

Design, synthesis, biological evaluation and docking studies of pterostilbene analogs inside PPARalpha.

Pterostilbene, a naturally occurring analog of resveratrol, has previously shown PPARalpha activation in H4IIEC3 cells and was found to decrease cholesterol levels in animals. In this study, analogs of pterostilbene were synthesized and their ability to activate PPARalpha was investigated. Among analogs that was synthesized (E)-4-(3,5-dimethoxystyryl)phenyl dihydrogen phosphate showed activity higher than pterostilbene and control drug ciprofibrate. Docking of the stilbenes inside PPARalpha showed the presence of important hydrogen bond interactions for PPARalpha activation.

Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats.

PURPOSE: Epidemiologic studies have linked the consumption of fruits and vegetables to reduced risk of several types of cancer. Laboratory animal model studies have provided evidence that stilbenes, phenolic compounds present in grapes and blueberries, play a role in inhibiting the risk of certain cancers. Pterostilbene, a naturally occurring stilbene from blueberries, was tested for its preventive activity against colon carcinogenesis. EXPERIMENTAL DESIGN: Experiments were designed to study the inhibitory effect of pterostilbene against the formation of azoxymethane-induced colonic aberrant crypt foci (ACF) preneoplastic lesions in male F344 rats. Beginning at 7 weeks of age, rats were treated with azoxymethane (15 mg/kg body weight s.c., once weekly for 2 weeks). One day after the second azoxymethane treatment, rats were fed experimental diets containing 0 or 40 ppm of pterostilbene. At 8 weeks after the second azoxymethane treatment, all rats were sacrificed, and colons were evaluated for ACF formation and for inhibition of inducible nitric oxide synthase (iNOS) and proliferating cell nuclear antigen. Effects on mucin MUC2 were also determined. RESULTS: Administration of pterostilbene for 8 weeks significantly suppressed azoxymethane-induced formation of ACF (57% inhibition, P < 0.001) and multiple clusters of aberrant crypts (29% inhibition, P < 0.01). Importantly, dietary pterostilbene also suppressed azoxymethane-induced colonic cell proliferation and iNOS expression. Inhibition of iNOS expression by pterostilbene was confirmed in cultured human colon cancer cells. CONCLUSIONS: The results of the present study suggest that pterostilbene, a compound present in blueberries, is of great interest for the prevention of colon cancer.

Methoxylation enhances stilbene bioactivity in Caenorhabditis elegans.

BACKGROUND: Stilbenes are 1,2-diphenylethylene congeners produced by plants in response to stress. Many stilbenes also exhibit xenobiotic activities in animal cells, such as inhibition of cancer cell growth, neuroprotection, and immune modulation. In vivo, hydroxylated stilbenes are metabolized by glucuronidation to facilitate excretion. Methoxylated stilbenes are metabolized more slowly, which may have a positive effect on in vivo bioactivity. Here, we have directly compared in vivo bioactivities of methoxylated and hydroxylated stilbenes in a whole organism using the roundworm Caenorhabditis elegans, an advantageous experimental system for such studies due to its rapid lifecycle, genetic amenability and relatively low-cost. RESULTS: Toxicity towards C. elegans adults was observed for trimethoxylated and dimethoxylated stilbenes, as well as the monomethoxylated stilbene desoxyrhapontigenin. Toxicity was not observed for the monomethoxylated stilbene, pinostilbene, nor for hydroxylated stilbenes. The methoxylated stilbenes that exhibited toxicity also showed stronger inhibitory effects than the hydroxylated stilbenes on germline tumor growth in gld-1(q485) adults. However, steady-state levels of three inhibitory methoxylated stilbenes did not directly correlate to their relative bioactivities. CONCLUSION: These findings demonstrate that, for the group of stilbenes investigated, methoxylation generally increased bioactivity in vivo in a whole organism, with the exception of pinostilbene. Differences in bioactivity in C. elegans adults did not appear to correlate with differential uptake. Rather, we speculate that methoxylated stilbenes may have increased interactions with biological targets in vivo or may interact with specific targets unaffected by hydroxylated stilbenes. The potent activities of methoxylated stilbenes provide a basis for further investigations to identify in vivo targets for these compounds.

Aminomethylphosphonic acid accumulation in plant species treated with glyphosate.

Aminomethylphosphonic acid (AMPA) is the most frequently detected metabolite of glyphosate in plants. The objective of this study was to determine if there is any correlation of metabolism of glyphosate to AMPA in different plant species and their natural level of resistance to glyphosate. Greenhouse studies were conducted to determine the glyphosate I 50 values (rate required to cause a 50% reduction in plant growth) and to quantify AMPA and shikimate concentrations in selected leguminous and nonleguminous species treated with glyphosate at respective I 50 rates. Coffee senna [ Cassia occidentalis (L.) Link] was the most sensitive ( I 50 = 75 g/ha) and hemp sesbania [ Sesbania herbacea (P.Mill.) McVaugh] was the most resistant ( I 50 = 456 g/ha) to glyphosate. Hemp sesbania was 6-fold and Illinois bundleflower [ Desmanthus illinoensis (Michx.) MacM. ex B.L.Robins. & Fern.] was 4-fold more resistant to glyphosate than coffee senna. Glyphosate was present in all plant species, and its concentration ranged from 0.308 to 38.7 microg/g of tissue. AMPA was present in all leguminous species studied except hemp sesbania. AMPA concentration ranged from 0.119 to 4.77 microg/g of tissue. Shikimate was present in all plant species treated with glyphosate, and levels ranged from 0.053 to 16.5 mg/g of tissue. Non-glyphosate-resistant (non-GR) soybean accumulated much higher shikimate than glyphosate-resistant (GR) soybean. Although some leguminous species were found to be more resistant to glyphosate than others, and there was considerable variation between species in the glyphosate to AMPA levels found, metabolism of glyphosate to AMPA did not appear to be a common factor in explaining natural resistance levels.