Influence of Carbohydrates on Secondary Metabolism in Fusarium avenaceum

Fusarium avenaceum is a widespread pathogen of important crops in the temperate climate zones that can produce many bioactive secondary metabolites, including moniliformin, fusarin C, antibiotic Y, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), chlamydosporol, aurofusarin and enniatins. Here, we examine the production of these secondary metabolites in response to cultivation on different carbon sources in order to gain insight into the regulation and production of secondary metabolites in F. avenaceum. Seven monosaccharides (arabinose, xylose, fructose, sorbose, galactose, mannose, glucose), five disaccharides (cellobiose, lactose, maltose, sucrose and trehalose) and three polysaccharides (dextrin, inulin and xylan) were used as substrates. Three F. avenaceum strains were used in the experiments. These were all able to grow and produce aurofusarin on the tested carbon sources. Moniliformin and enniatins were produced on all carbon types, except on lactose, which suggest a common conserved regulation mechanism. Differences in the strains was observed for production of fusarin C, 2-AOD-3-ol, chlamydosporol and antibiotic Y, which suggests that carbon source plays a role in the regulation of their biosynthesis.     

Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fractionation

The cytotoxicity of extracts from rice cultures of five Fusarium avenaceum strains against the porcine epithelial kidney cell-line PK-15 was investigated using the Alamar Blue™ assay. After the identification of known fungal metabolites, cytotoxic extracts were fractionated using semi-preparative reversed-phase HPLC and normal phase LC, and the fractions were tested for cytotoxicity. In this way, two different groups of metabolites were identified as the major cytotoxic principles of the extracts. High concentrations of enniatins, especially enniatins B and B1, inhibited the metabolic activity of PK-15 cells. Furthermore, an unidentified metabolite, produced in high amounts by a strain that produced relatively small amounts of enniatins, was also found to be cytotoxic to PK-15 cells. This study shows that enniatins, a group of cyclic depsipeptides, which have been ignored as significant contributors to the toxicity of fungal extracts, may account for most of the observed effect for F. avenaceum.     

Cytotoxicity of enniatins A, A1, B, B1, B2 and B3 from Fusarium avenaceum

The enniatins A, A1, B, B1, B2 and B3 were purified from hexane-extracts of Fusarium avenaceum rice cultures, using semi-preparative HPLC, after precipitation of lipids. Their toxicity, as well as the toxicity of the related fungal metabolite beauvericin (Bea) and the trichothecenes deoxynivalenol (DON) and T-2 toxin, was tested in two cell lines of human origin (hepatocellular carcinoma-line Hep G2 and fibroblast-like foetal lung cell line MRC-5) by using the BrdU and Alamar Blue™ assays. All the compounds evoked toxicity in the in vitro assays at the concentrations tested. The MRC-5 cell line in combination with the BrdU assay resulted in the lowest inhibitory concentrations (IC₅₀) for exposure with enniatins in the range 0.8 μM (enniatin A) to 3.6 μM (enniatin B). The cytotoxicity of DON in the BrdU assay was comparable to the cytotoxicity of enniatins A, B and Bea in a multiple regression model, while DON was significantly more cytotoxic than the enniatins in the Alamar Blue™ assay. This study indicates that enniatins, fungal metabolites that are commonly found in grain in Northern Europe, may have an underestimated toxic potential.     

2-Amino-14,16-dimethyloctadecan-3-ol, a new sphingosine analogue toxin in the fungal genus Fusarium

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) was isolated from the cytotoxic rice culture extract of a strain of Fusarium avenaceum, which had previously been isolated from Norwegian grain. The structural information was obtained from LC-MS/MS, GC-MS, NMR spectroscopy and high-resolution MS data. The metabolite has a striking similarity to sphinganine, an intermediate in the biosynthesis of the sphingolipids. This similarity is a major feature of the so-called sphingosine analogue toxins; the most studied being the AAL toxins and the fumonisins. 2-AOD-3-ol was found to be cytotoxic to the rat hepatoma cell line H4IIE-W and to the porcine epithelial kidney cell line PK(15) at concentrations (EC₅₀) of 16 and 24 μM, respectively. The metabolite has been found in F. avenaceum inoculated wheat that was treated to support ideal conditions for Fusarium growth, demonstrating that the fungus has the potential to produce the metabolite under field conditions, which may occur in Northern Europe.     

植物内生真菌Talaromyces sp.的次级代谢产物研究

摘要：目的 对1株植物内生真菌Talaromyces sp.的次级代谢产物进行研究。方法 采用稻米培养基培养,通过正反相 硅胶柱层析、Sephadex LH-20凝胶柱层析、半制备反相高效液相色谱等技术对发酵提取物进行分离纯化,运用质谱、核磁 共振、圆二色谱等波谱技术进行结构鉴定,采用MTT测定化合物的细胞毒活性。结果 从该菌株中共分离鉴定11个已知化 合物,其中包括9个聚酮类化合物(1~9)和2个环肽类化合物(10和11),分别为1-deoxyrubralactone(1)、alternariol(2)、alternariol 5-methyl ether(3)、altenuisol(4)、3-hydroxyalternariol 5-O-methyl ether(5)、(2'R,4'R,5'R)-altenuene(6)、(2'S,4'R,5'R)-isoaltenuene(7)、 (2'R,4'R,5'R)-altenuene-5'-acetoxyester(8)、7-hydroxy-3,5-dimethyl-isochromen-1-one(9)、腾毒素(10)和二氢腾毒素(11)。结论 化合 物1、化合物3~11为首次从该属菌株中分离,化合物2和3在100 μmol/L的浓度下对人膀胱癌J82细胞株具有抑制活性,抑制率分 别为56.9%和59.7%。     

Two new cytotoxic quinone type compounds from the halotolerant fungus Aspergillus variecolor

Two new quinone type compounds, variecolorquinones A (1) and B (2) together with eleven known related compounds 3 approximately 13 have been isolated from the metabolites produced by the halotolerant fungal strain Aspergillus variecolor B-17. The structures of 1 and 2 were determined by spectroscopic methods. 1 exhibited selective cytotoxicity against A-549 cells with the IC50 values of 3.0 microM. 2 showed cytotoxicity against HL60 and P388 cells with the IC50 values of 1.3 and 3.7 microM, respectively.     

中国南海聚裂丛柳珊瑚化学成分研究

目的研究聚裂丛柳珊瑚Rumphella aggregata的化学成分。方法利用硅胶色谱、Sephadex LH-20凝胶色谱、HPLC等手段对化学成分进行分离纯化;通过理化性质、波谱分析方法结合文献对照,鉴定化合物的结构。结果从聚裂丛柳珊瑚甲醇提取物中,共分离鉴定了15个单体化合物:胆甾醇(1)、(22E)-胆甾-5,22-二烯-3β-醇(2)、(22E)-麦角甾-5,22-二烯-3β-醇(3)、麦角甾-5,24(28)-二烯-3β-醇(4)、豆甾-5,24(28)-二烯-3β-醇(5)、柳珊瑚甾醇(6)、胆甾-7-烯-3β,5α,6β-三醇(7)、(22E)-胆甾-7,22-二烯-3β,5α,6β-三醇(8)、麦角甾-7-烯-3β,5α,6β-三醇(9)、(22E)-麦角甾-7,22-二烯-3β,5α,6β-三醇(10)、豆甾-7-烯-3β,5α,6β-三醇(11)、(22E)-豆甾-7,22-二烯-3β,5α,6β-三醇(12)、尿嘧啶(13)、咖啡因(14)、hydratoperidinin(15)。结论化合物1～15均为首次从该属柳珊瑚中分离得到,并首次对化合物hydratoperidinin(15)的1HNMR及13 CNMR信号进行了全归属。化合物12在10μg.mL-1浓度水平,对K562肿瘤细胞株的抑制率为39.17%。     

Extracts from two marine sponges lower cyclin B1 levels, cause a G2/M cell cycle block and trigger apoptosis in SW-13 human adrenal carcinoma cells.

Marine sponges have been shown to produce metabolites with cell growth- and endocrine-altering activities. We tested extracts from two species: the 'brown variable sponge' (Anthosigmella varians) and the 'West Indian bath sponge' (Spongia barbara), for effects on the cell cycle regulatory protein, cyclin B1; cell cycle growth-phase (sub-G1/apoptosis, G1, S, and G2/M); and cell survival in SW-13 human adrenal carcinoma cultures. Polyacrylamide gel electrophoresis studies indicated a 70-90% reduction in cyclin B1 levels by treatment with these agents. Microscopic examination of cultures with DAPI staining showed dense and fragmented DNA fluorescence, characteristic of apoptosis, in both sponge extract-treated cultures but not in controls. Flow cytometry analysis showed a 16-fold increase in the percentage of cells entering apoptosis (sub-G1 phase of cell cycle) by treatment with Anthosigmella varians extract (p <0.01) and a 10-fold increase using Spongia barbara extract (p <0.01) During this same time, the percentage of cells in G2/M was increased 1.6-fold by Anthosigmella varians extract (p <0.01) and 2.0-fold by Spongia barbara extract (p <0.01) Cell growth/survival studies also indicated a time-dependent decline in the percentage confluence of cell cultures exposed to Anthosigmella varians or Spongia barbara extracts. These experiments demonstrate that some species of marine sponges have metabolites which are capable of interfering with the mammalian cell cycle and with the survival of human adrenal carcinoma cells in culture.     

Microbial fermentation-derived inhibitors of efflux-pump-mediated drug resistance

A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa and Candida albicans. New compounds EA-371alpha and EA-371delta were isolated and demonstrated to be potent and specific inhibitors of the MexAB-OprM pump in P. aeruginosa. Two series of fungal metabolites, enniatins and beauvericins, were found to be ubiquitous and potent inhibitors of ABC transporters. Milbemycins were rediscovered as potent inhibitors of the CDRI pump in C. albicans, and demonstrated to potentiate effectively the antifungal activity of fluconazole and SCH-56592 against a wide variety of Candida clinical isolates.     

Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria

Background

Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions.

Results

All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF-12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT).

Conclusions

Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption.     

中国南海短指软珊瑚化学成分研究

目的研究短指软珊瑚Sinulariasp.的化学成分。方法利用硅胶色谱、Sephadex LH-20凝胶色谱、HPLC等手段对化学成分进行分离纯化;通过理化性质、波谱分析方法结合文献对照,鉴定化合物的结构。结果从短指软珊瑚甲醇提取物中,共分离鉴定了11个单体化合物:胆甾醇(1)、(22E)-麦角甾-5,22-二烯-3β-醇(2)、胆甾-5,20-二烯-3β-醇(3)、麦角甾-5,24(28)-二烯-3β-醇(4)、柳珊瑚甾醇(5)、3β-羟基胆甾-5-烯-7-酮(6)、3β-羟基麦角甾-5,24(28)-二烯-7-酮(7)、(22E)-3β-羟基麦角甾-5,22-二烯-7-酮(8)、3β-羟基麦角甾-5-烯-7-酮(9)、(22E)-3β-羟基胆甾-5,22-二烯-7-酮(10)、鲨肝醇(11)。结论化合物6～10为首次从该属软珊瑚中分离得到。化合物7在10μg.mL-1浓度水平,对K562肿瘤细胞株的抑制率为22.74%,对HeLa肿瘤细胞株的抑制率为9.98%。     

A Strategy for Quality Control of Menispermum dauricum DC Based on Cytotoxic Activity and HPLC Fingerprint Analysis

The rhizome of Menispermum dauricum DC known as a traditional Chinese medicine, with high content of alkaloids, has been found to possess antitumor activity. In this research, an attempt to correlate fingerprinting with bioactivity was made for quality control of M. dauricum. Firstly, the cytotoxicity of extracts from ten batches of samples against human breast MCF-7 cancer cells was estimated by [3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazoliumbromide] assay. Then, cytotoxic activity-integrated fingerprints were established by high performance liquid chromatography. Eight peaks were selected as the common peaks to evaluate the similarities of samples and hierarchical clustering analysis was used to identify and classify different samples into groups. Assays for determinations of total alkaloids and dauricine contents enabled cytotoxicity coefficient of each extract. The potential usefulness of employing cytotoxicity coefficient was investigated by a combination of Pearson correlation coefficients and multiple linear regression analysis as being the reliable parameter to evaluate the herbal extracts. The results indicated that the level of dauricine (peak 8 in the fingerprint) correlated closely with cytotoxicity and played a significant role in the cytotoxicity of Bei Dou-Gen and could be related to its antitumor properties. It is proposed that the cytotoxicity coefficient value with a cytotoxic activity-integrated fingerprint of key biomarkers (dauricine) may be useful indicators to adopt for the quality control of M. dauricum. The analysis of cytotoxic-activity-integrated fingerprint could correlate fingerprinting with bioactivities and would provide a reasonable strategy for quality control of complex mixture of herbal medicines.     

Cytotoxicity/genotoxicity: The application of cell culture techniques to the measurement of marine sediment pollution

Fish cell cultures were used to determine whether in vitro aquatic animal cell culture systems were capable of detecting pollution in the marine environment. Cells derived from rainbow trout gonad (RTG-2) and bluegill fry tissues (BF-2) were used as model cell systems to measure cytotoxicity and genotoxicity following exposure to Puget Sound sediment extracts, benzo(a)pyrene and MNNG. Sediment was collected from several sites within Puget Sound known to be contaminated with compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorinated hydrocarbons and heavy metals. Each of the sediment samples was extracted with organic solvents and added to cultures of the two model cell systems in DMSO. Following exposure the cultures were evaluated for cell death, mitotic inhibition, stimulatory effects, and chromosomal damage. These cell cultures responded to the sediment extracts much as they did to known mutagenic/carcinogenic chemicals which were used as model compounds. Those sediments known to be contaminated, based on chemical analysis and historical use patterns were also found to be qualitatively and quantitatively more toxic than were sediments from areas which were relatively unaffected by human activity. The results show that in vitro cell systems are capable of detecting pollution in the marine environment and have the potential of being powerful tools in aquatic toxicology in conjunction with whole animals or as a method of screening materials prior to in vivo testing.     

Identification of inhibitors of inducible nitric oxide synthase from microbial extracts

A new member of the angucycline family, vineomycin C (3), together with four known metabolites saquayamycin A1 (1), A-7884 (2), rabelomycin (5) and xanthomegnin (6) were isolated from microbial extracts. The structures were determined by 1D and 2D NMR techniques and chemical degradation. Compounds 1-3 and 5 were isolated from a fermentation of Streptomyces sp., while 6 was isolated from a fungal fermentation extract. All five compounds have shown potent inhibitory activity in the inducible nitric oxide synthase (iNOS) assay.     

Metabolism and cytotoxicity of 7,12-dimethylbenz[a]anthracene by hamster, rat and rabbit embryo cell cultures.

1. The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by rat, hamster and rabbit fibroblasts has been studied and compared with the metabolism of the hydrocarbon by liver homogenates. The metabolsim of DMBA by cell cultures and by liver homogenates was very similar. The ethyl acetate-soluble metabolites identified included dihydrodiols, phenols and hydroxymethyl derivatives. Other more polar metabolites and unidentified water-soluble metabolites were also formed. 2. High yields of phenols and other more polar metabolites, which may be tetrahydrotetrols, were produced by rabbit fibroblasts. 3. Kinetic studies showed that the metabolic activity of rabbit fibroblasts was high but that the conversion of DMBA into water-soluble metabolites was lower than with hamster and rat fibroblasts. 4. 7,12-Dimethylbenz[a]anthracene-induced cytotoxicity was inversely related to the conversion of metabolized DMBA to water-soluble derivatives.     

The nature of halogen substitution determines the mode of cytotoxicity of halopropanols

The cytochrome P450-dependent generation of reactive metabolites from 1,3-dichloropropanol and 1,3-dibromopropanol was assessed in a microsomal thiol depletion assay, while the toxicity of these compounds was assessed in rat hepatocyte cultures and in the 3T3 cell line. Thiol-depleting metabolites of both compounds were generated in the microsomal assay; however, only dibromopropanol extensively depleted glutathione when glutathione S-transferase was used as the enzyme source. The cytotoxicity of dichloropropanol was both cytochrome P450- and glutathione-dependent, whereas that of dibromopropanol was glutathione-dependent but largely independent of cytochrome P450. These results indicate that the mechanisms underlying the cytotoxicity of halopropanols are dependent on the nature of the halogen substitution and that microsomal and cellular assays for reactive metabolite generation may yield conflicting results.     

Antibacterial activity and in vitro cytotoxicity of extracts and fractions of Parkia biglobosa (Jacq.) Benth. stem bark and Ageratum conyzoides Linn. leaves

Many species of plants in African countries are widely used in the rural communities where there is little or no access to modern medicine. However, the safety and effectiveness of these medicinal plants are poorly evaluated. The stem bark of Parkia biglobosa Jacq. and leaves of Ageratum conyzoides Linn. were investigated for their antibacterial and cytotoxic activities. The plant materials were extracted with 95% ethanol, and fractionated with petroleum ether, chloroform and ethyl acetate. The antibacterial effects of the extracts and fractions of the plant materials were assayed on the bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA) and Clostridium perfringes. Ethanol extracts of P. biglobosa and A. conyzoides were screened for cytotoxicity using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Two cancer cell lines (SK-MES 1 and SK-LU 1) and one normal cell line (human skin fibroblast cell line, FS5) were used for the screening of the extracts and the fractions obtained. The ethanolic extracts and fractions of P. biglobosa and A. conyzoides showed the best activity against E. coli, S. aureus and MRSA. All fractions of A. conyzoides leaves have no activity against P. aeruginosa. Human lung cancer cell lines (SK-LU 1 and SK-MES 1) and human skin fibroblast cell line (FS5 cells) were treated with various concentrations (3.9μg/ml-2mg/ml) of the extracts and fractions for 24h. SK-MES 1 cells are more susceptible to treatment with the plant fractions. All the fractions of A. conyzoides leaves and the petroleum ether fraction of P. biglobosa were cytotoxic to SK-MES 1 cells, which to some extent may support their traditional inclusion in herbal preparations for treatment of cancer. The overall results provided evidence that the studied plant extracts might be potential sources of new antibacterial and anticancer drug.     

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Metabolism of atenolol in man.

1. The disposition and metabolism of 1-(4-carbamoyl[14C]methylphenoxy)-3-isopropylaminopan-2-ol (atenolol, Tenormin) has been studied in man following oral and intravenous doses. 2. Approx. 50% of an oral dose was eliminated in urine; the major radiolabelled component was atenolol (approx. 90%). Faecal extracts also contained largely unchanged atenolol, with small amounts of more polar metabolites. Biliary excretion of atenolol and its metabolites is not a major route of elimination in man. Metabolism of the compound is not extensive and route-dependent modes of metabolism do not appear to complicate the position. 3. Atenolol appeared to be the only major radiolabelled component in blood. 4. Oral doses of atenolol are incompletely absorbed (range 46-62%), even when formulated as a solution. 5. 1-[4-(C-Carbamoylhydroxymethyl)phenoxy]-3-isopropylaminopropan-2-ol was a minor urinary metabolite, which has only one tenth the activity of the parent compound as a beta-adrenergic blocking agent in the rat. 6. Pharmacological activity in man appears to be due to atenolol alone.     

Effects of altering the electronics of 2-methoxyestradiol on cell proliferation, on cytotoxicity in human cancer cell cultures, and on tubulin polymerization

A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the relationships between structure and activity. The compounds were designed to diminish the potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of tubulin polymerization and for cytotoxicity. 17alpha-methyl-beta-estradiol (30), 2-propynyl-17alpha-methylestradiol (39), 2-ethoxy-17-(1'-methylene)estra-1,3,5(10)-triene-3-ol (50) and 2-ethoxy-17alpha-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than 2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating metabolic processes. All of the compounds tested were cytotoxic in the panel of 55 human cancer cell cultures, and generally, the derivatives that displayed the most activity against tubulin were also the most cytotoxic.