A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Beauvericin cytotoxicity to the invertebrate cell line SF-9

The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM, for different periods of time (from 30' to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 microM concentration. This effect became gradually more evident at higher concentrations (approximately equal to 28% at 30 microM, approximately equal to 50% at 100 microM, approximately equal to 68% at 300 microM). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 microM beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations approximately equal to 23% at 3 microM, approximately equal to 47% at 10 microM, approximately equal to 65% at 30 microM, approximately equal to 90% at 100 microM, approximately equal to 99% at 300 microM). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 microM and 10 microM, respectively. In time-course experiments, no effect of beauvericin (30 microM) on cell viability could be seen after exposure for periods of time as long as 30', 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 microM. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.     

Cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin B1 in the SF-9 insect cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum

Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties.The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50% of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were > or =2.07 +/- 0.08 microM for FRhK-4 cells: > or =2.23 +/- 0.11 microM for Vero cells; > or =2.54 +/- 0.18 microM for MA104 cells; and > or =2.70 +/- 0.20 microM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 +/- 0.68%, 14.48 +/- 5.06% and 21.47 +/- 3.74% for HSV-1 (strain KOS); 5.96 +/- 2.51%, 8.75 +/- 3.08% and 17.75 +/- 5.19% for HSV-1 (strain ATCC/VR-733); 5.13 +/- 2.38 %, 8.18 +/- 1.11% and 8.51 +/- 1.94% for poliovirus type 2; 8.30 +/- 4.24%; 13.33 +/- 4.66% and 24.27 +/- 2.18% for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay.     

The emerging <Emphasis Type="Italic">Fusarium</Emphasis> toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC₂₀ and IC₅₀ values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 μM and 4 μM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC₂₀ 0.7 μM, IC₅₀ of 0.8 μM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations.     

Identification and heritability of fumonisin insensitivity in Zea mays

Landraces of maize (Zea mays ssp. mays) and its wild teosinte relatives (Zea mays spp. parviglumis and mexicana) were surveyed for sensitivity to fumonisin B(1), a phytotoxin produced by the maize pathogen Gibberella moniliformis. Only two of 42 Z. mays samples were highly insensitive to FB(1) (ED(50) = ca. 200 microM). The teosintes and 76% of the maize landraces were moderately or highly sensitive to FB(1) (ED(50) < or = 30 microM), which indicates that FB(1) sensitivity is likely to be an ancestral trait in Z. mays. F(1) generations derived from crosses between FB(1)-sensitive maize inbred B73 and insensitive landraces were significantly less sensitive than B73. Thus, our data indicate that FB(1)-insensitivity is a relatively rare but heritable trait in maize. We also report the sensitivity of maize to other Gibberella toxins - beauvericin, diacetoxyscirpenol, and moniliformin.     

Simultaneous determination of type A-& B-trichothecenes and zearalenone in cereals by High Performance Liquid Chromatography — Tandem Mass Spectrometry

A rapid quantitative method for the simultaneous determination of the majorFusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize and wheat was developed. Raw extracts (acetonitrile/water 84/16) are cleaned-up with MycoSep^® columns., Chromatographic separation and end determination is carried out by HPLC-APCI-MS/MS.HPLC run times of 10 minutes considerably increases sample throughput and make this method suitable for routine analysis. The use of a triple quadrupole mass spectrometer allows the selective detection of the mycotoxins and their quantification in the low μg/kg-range.     

A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B₁, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B₁ and M₁ be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.     

Mycotoxins and fungi in wheat harvested during 1990 in test plots in the state of São Paulo,Brazil

Wheat from two cultivars with contrasting characteristics were harvested in ten experimental plots located in wheat producing areas of the State of São Paulo, Brazil. The samples (10 of each cultivar) were analyzed by a gaschromatographic method for deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), toxins T-2 (T-2) and HT-2, T-2 tetraol, T-2 triol, and by a thin-layer chromatographic method for zearalenone (ZEN), aflatoxins B₁, B₂, G₁, G₂, ochratoxin A and sterigmatocystin. No mycotoxins were detected in 13 samples. DON was found in four samples (0.47–0.59 µg/g), NIV in three samples (0.16–0.40 µg/g), T-2 in two samples (0.40, 0.80 µg/g), DAS in one sample (0.60 µg/g), and ZEN in three samples (0.04–0.21 µg/g). The wheat samples were also examined for the incidence of fungi.Alternaria, Drechslera, Epicoccum andCladosporium were the prevailing genera. Among theFusarium spp.,F. semitectum was present in 19 samples andF. moniliforme in 18 samples. NoF. graminearum was isolated in the samples.Abbreviations DAS diacetoxyscirpenol - DON deoxynivalenol - NIV nivalenol - T-2 T-2 toxin - ZEN zearalenone     

Determination of B-trichothecenes in wheat by post column derivatisation liquid chromatography with fluorescence detection (PCD-HPLC-FLD)

B-trichothecenes are one of the most common contaminants of cereals in Europe. Therefore, the use of fast and accurate methods is necessary to measure contamination levels and observe regulatory limits. At the moment, mostly gas chromatographic (GC) methods are used but HPLC-UV methods are also employed. Clean-up is commonly done either with immunoaffinity or Mycosep® columns. In the Christian Doppler Laboratory for Mycotoxin Research we have established an alternative HPLC method with post column derivatisation (PCD) as an alternative to existing chromatographic methods. This PCD-HPLC-FLD method uses a Mycosep® clean-up and allows the simultaneous detection and quantification of deoxynivalenol, nivalenol, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. A validation with wheat gave for deoxynivalenol a limit of quantification ten times below the drafted European Union guideline level (500 µg.kg^?1) and a limit of detection of 8 µg.kg^?1. The relative standard derivation for DON was 10% (n=30). The obtained mean recovery rate for DON was 90% in a range from 50 to 1000 µg.kg^?1.     

The hypolipidemic action of antibiotic 86/88 (enniatin B) in a hepatoblastoma G2 cell culture]

A cyclodepsipeptide antibiotic 86/88 (enniatin B) with strong hypolipidemic action was isolated from the culture liquid of the fungus INA F-86/88 identified as Fusarium lateritium Nees var. stilboides (Wr.) Bilai. In the Hep G2 cell culture the antibiotic suppressed 14C-acetate incorporation into cholesterol (IC50 1.75 microM), cholesterol ethers (IC50 1 microM), triglycerides (IC50 1.3 microM) and free fatty acids (IC50 2.2 microM). The most pronounced effect of the drugs, i.e. the suppression of the cholesterol ethers synthesis is likely due not only to the ACAT inhibition but also to the inhibition of the triglyceride synthesis and the diminishing of the free fatty acids pool in the cells.     

Development,validation and application of a multi-mycotoxin method for the analysis of whole wheat plants

Mycotoxins are known to affect the health of humans and husbandry animals. In contrast to wheat grains used for food and feed, whole wheat plants are rarely analysed for mycotoxins, although contaminated straw could additionally expose animals to these toxic compounds. Since the entire wheat plant may also act as source of mycotoxins emitted into the environment, an analytical method was developed, optimised and validated for the analysis of 28 different mycotoxins in above-ground material from whole wheat plants. The method comprises solid-liquid extraction and a clean-up step using a Varian Bond Elut Mycotoxin^® cartridge, followed by liquid chromatography with electrospray ionisation and triple quadrupole mass spectrometry. Total method recoveries for 26 out of 28 compounds were between 69 and 122% and showed limits of detection from 1 to 26 ng/g_{dry weight (dw)}. The overall repeatability for all validated compounds was on average 7%, and their mean ion suppression 65%. Those rather high matrix effects made it necessary to use matrix-matched calibrations to quantify mycotoxins within whole wheat plants. The applicability of this method is illustrated with data from a winter wheat test field to examine the risks of environmental contamination by toxins following artificial inoculation separately with four different Fusarium species. The selected data originate from samples of a part of the field which was inoculated with Fusarium crookwellense. In the wheat samples, various trichothecenes (3-acetyl-deoxynivalenol, deoxynivalenol, diacetoxyscirpenol, fusarenone-X, nivalenol, HT-2 toxin, and T-2 toxin) as well as beauvericin and zearalenone were identified with concentrations ranging from 32 ng/g_dw to 12 × 10³ ng/g_dw.     

Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were determined by enzyme-linked immunosorbent assay. The following mycotoxins were found in these lichens in a broad concentration range with a frequency of 70–100%: sterigmatocystin (7–2090 ng/g), alternariol (20–6460 ng/g), and emodin (45–94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B₁ was detected in only six species with a frequency of 2–42%, whereas roridin A was present in 10% of Hypogymnia physodes samples.     

Moniliformin and the European Corn Borer (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>)

A representative survey was made of maize ears of the 1988 and 1989 crop in Austria to establish the influence of corn borer injuries onFusarium species involved in ear fusariosis andFusarium toxin production.TheFusarium species most frequently isolated from rot-damaged ears wereFsacchari var. subglutinans (about 50 %) andF. graminearum (about 30 %). There was a striking difference between theFusarium species of the Liseola and the Discolor section concerning their occurrence on corn borer-damaged ears. More than 80 % of the ears infected withF. sacchari var. subglutinans andF. verticillioides, but less than 15 % of the ears infected withF. graminearum, F. crookwellense andF. culmorum showed corn borer injuries.Toxin analyses of the infected ears corresponded to the known toxigenicity of the respectiveFusarium species. Ears infected withF. sacchari var. subglutinans contained moniliformin (up to 20 mg/kg), those infected withF. verticillioides fumonisin B₁ and B₂ (up to 15 mg/kg). In ears infected withF. graminearum, F. culmorum andF. crookwellense zearalenone (up to 40 mg/kg) and deoxynivalenol (up to 500 mg/kg) or nivalenol (up to 10 mg/kg), respectively, could be detected. Hence measures to combat the European corn borer will mainly reduce moniliformin and fumonisin contamination, but will affect zearalenone, deoxynivalenol and nivalenol contents of the ears to a much lesser extent.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

Enniatin has a new function as an inhibitor of Pdr5p, one of the ABC transporters in Saccharomyces cerevisiae

Pdr5p is one of the major multidrug efflux pumps whose overexpression confers multidrug resistance (MDR) in Saccharomyces cerevisiae. By using our original assay system, a fungal strain producing inhibitors for Pdr5p was obtained and classified as Fusarium sp. Y-53. The purified inhibitors were identified as ionophore antibiotics, enniatin B, B1, and D, respectively. A non-toxic concentration of each enniatin (5 microg/ml, approximately 7.8 microM) strongly inhibited a Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. The enniatins accumulated a fluorescent dye rhodamine 123, a substrate of Pdr5p, into yeast cells. The mode of Pdr5p inhibition of enniatin was competitive against FK506, and its inhibitory activity was more potent with less toxicity than that of FK506. The enniatins showed similar inhibitory profile as FK506 against S1360 mutants (S1360A and S1360F) of Pdr5p. The enniatins did not inhibit the function of Snq2p, a homologue of Pdr5p. Thus, it was found that enniatins are potent and specific inhibitors for Pdr5p, with less toxicities than that of FK506.