Identification of Volatile Sulfur Compounds Produced by Schizophyllum commune.

Schizophyllum commune is a causative agent of allergic bronchopulmonary mycosis, allergic fungal rhinosinusitis, and basidiomycosis. Diagnosis of these diseases remains difficult because no commercially available tool exists to identify the pathogen. Unique volatile organic compounds produced by a pathogen might be useful for non-invasive diagnosis. Here, we explored microbial volatile organic compounds produced by S. commune. Volatile sulfur compounds, dimethyl disulfide (48 of 49 strains) and methyl ethyl disulfide (49 of 49 strains), diethyl disulfide (34 of 49 strains), dimethyl trisulfide (40 of 49 strains), and dimethyl tetrasulfide (32 of 49 strains) were detected from headspace air in S. commune cultured vials. Every S. commune strain produced at least one volatile sulfur compound analyzed in this study. Those volatile sulfur compounds were not detected from the cultures of Aspergillus spp. (A. fumigatus, A. flavus, A. niger, and A. terreus), which are other major causative agents of allergic bronchopulmonary mycosis. The last, we examined H2S detection using lead acetate paper. Headspace air from S. commune rapidly turned the lead acetate paper black. These results suggest that those volatile sulfur compounds are potent targets for the diagnosis of S. commune and infectious diseases.

Non-target environmental analysis by liquid chromatography/high-resolution mass spectrometry with a product ion and neutral loss database.

Despite the increasing detection of emerging substances in the environment, the identity of most are left unknown due to the lack of efficient identification methods. We developed a non-target analysis method for identifying unknown substances in the environment by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) with a product ion and neutral loss database (PNDB). The present analysis describes an elucidation method with elemental compositions of the molecules, product ions, and corresponding neutral losses of the unknown substance: (1) with the molecular formula, possible molecular structures are retrieved from two chemical structure databases (PubChem and ChemSpider); then (2) with the elemental compositions of product ions and neutral losses, possible partial structures are retrieved from the PNDB; and finally, (3) molecular structures that match the possible partial structures are listed in order of number of hits. A molecular structure with a higher number of hits is more similar to the structure of the analyzed substance. The performance of the non-target method was evaluated by simulated analysis of 150 LC/HRMS spectra registered in MassBank. First, all substances of the same mass data (41/41) and 68% (39/57) of the mass data of the same substances not registered in the PNDB were elucidated. It was demonstrated that 14% (7/52) and 31% (16/52) of the substances with no mass spectral data registered in the PNDB were obtained at the first and within the fifth place, respectively. Owing to the fact that 10 of the total hits occurred in product ions and neutral losses, almost 50% of the substances evaluated with this method were placed at the top 4 positions in the similarity ranking. Importantly, the proposed method is effective for analyzing mass spectral data that has not been registered in the PNDB and thus is expected to be used for a variety of non-target analyses.

Microflora of Mycotoxigenic Fungi in Rice Grains in Kyushu Region of Japan and Their Changes during Storage under non-Controlled Conditions.

Contamination of agricultural crops by mycotoxins has increased because of the expansion of mycotoxin-producing fungi along with global warming. In this study, the fungal microflora of brown rice grains cultivated in Kyushu region in the southern part of Japan was investigated. A total of 75% of rice samples examined in this study showed less than 30% of fungal contamination rates with a median rate of 12.5%. Some isolates of Aspergillus flavus showed the ability to produce aflatoxins (AFs) (AFB1 production was 62.5-70.4 ng/mL) . Furthermore, AF-producing Aspergillus flavus survived during storage and Aspergillus creber, which produced sterigmatocystin, was detected in a stored rice sample. Although AFs or sterigmatocystin-contamination was not detected in any rice samples, these mycotoxin-producing fungi are distributed and can survive during storage under the natural conditions in Japan. Employing suitable storage conditions is important for preventing mycotoxin contamination of brown rice grains.

The In Vivo and In Vitro Toxicokinetics of Citreoviridin Extracted from Penicillium citreonigrum.

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi; however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.

Comparative genome analysis of Aspergillus flavus clinically isolated in Japan.

Aspergillus flavus is an important zoonotic pathogen and a well-known aflatoxin producer. Aspergillus flavus strains that are prevalent in Japanese environments are reported to be non-aflatoxigenic, although their aflatoxin productivity, especially among clinical isolates, has not been thoroughly investigated to date. In this study, we sequenced the genomes of ten strains of A. flavus isolated in Japan and compared their sequences with each other as well as with those of Aspergillus oryzae RIB40 and A. flavus NRRL3357. The phylogenetic analysis based on identified SNPs indicated that five strains were closer to A. oryzae RIB40 than to A. flavus NRRL3357. In contrast, of those isolates that were closer to A. flavus NRRL3357 than to A. oryzae RIB40, three were found to possess either the entire or partial aflatoxin biosynthesis gene cluster of NRRL3357-type. Furthermore, two of the three actually produced either aflatoxin B1 or an intermediate of the reaction leading to aflatoxin formation. Three of the ten strains we isolated were identified to possess part of the aflatoxin gene cluster, while five others retained the A. oryzae RIB40-type cluster. The genome data thus obtained may be further explored and utilized for comparative analysis of aflatoxin production in environmental and clinical isolates of A. flavus.

Developmental exposure of citreoviridin transiently affects hippocampal neurogenesis targeting multiple regulatory functions in mice.

To investigate the developmental exposure effect of citreoviridin (CIT) on postnatal hippocampal neurogenesis, pregnant ICR mice were dietary exposed to CIT at 0, 1, 3 and 10 ppm from gestation day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without CIT exposure. Male offspring were analyzed. At 10 ppm on PND 21, weak changes suggestive of neural stem cell reduction and progenitor cell proliferation were observed. Number of hilar CALB1+ interneurons reduced, suggesting an influence on neurogenesis. In contrast, number of hilar SST+ interneurons increased and Bdnf and Ntrk2 transcripts upregulated in the dentate gyrus, suggesting a facilitation of BDNF-TRKB signaling for progenitor cell proliferation. Transcript expression changes of an outside regulatory system suggested suppressed function of GABAergic interneurons, especially of PVALB+ interneurons for compensation on neural stem cell reduction. At ≥ 3 ppm, number of ARC+ mature granule cells increased, and at 10 ppm, number of hilar GRIA1+ cells increased and Gria2 and Gria3 upregulated, suggesting an operation of AMPA receptor membrane trafficking on the increase of ARC-mediated synaptic plasticity. On PND 77, all the transcript expression changes of the neurogenesis regulatory system except for Grin2d were inverted, suggesting an operation of a homeostatic mechanism on CIT-induced disruptive neurogenesis. Simultaneous downregulation of Grin2a and Grin2d suggests suppression of GABAergic interneuron function to adjust neurogenesis at the normal level. The no-observed-adverse-effect level of CIT for offspring neurogenesis was determined to be 1 ppm, translating to 0.13-0.51 mg/kg body weight/day of maternal oral exposure.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 µg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

GliA in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin.

Gliotoxin is an important virulence factor of Aspergillus fumigatus. Although GliA putatively belongs to the major facilitator superfamily in the gliotoxin biosynthesis cluster, its roles remain unclear. To determine the function of GliA, we disrupted gliA in A. fumigatus. gliA disruption increased the susceptibility of A. fumigatus to gliotoxin. The gliT and gliA double-disrupted mutant had even higher susceptibility to gliotoxin than each individual disruptant. The extracellular release of gliotoxin was greatly decreased in the gliA disruptant. Mice infected with the gliA disruptant of A. fumigatus showed higher survival rates than those infected with the parent strain. These results strongly indicate that GliA, in addition to GliT, plays a significant role in the tolerance to gliotoxin and protection from extracellular gliotoxin in A. fumigatus by exporting the toxin. This also allows the fungus to evade the harmful effect of its own gliotoxin production. Moreover, GliA contributes to the virulence of A. fumigatus through gliotoxin secretion.

[Latest development in mass spectrometry for clinical application].

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review summarizes the state of the art in mass spectrometry and related techniques for clinical application with a main focus on recent developments in LC-MS. Current trends in ionization techniques, automated online sample preparation techniques coupled with LC-MS, and ion mobility spectrometry are discussed. Emerging mass spectrometric approaches complementary to LC-MS are discussed as well.

Analysis of tolfenpyrad and its metabolites in plasma in a tolfenpyrad poisoning case.

Tolfenpyrad (TFP) is a pesticide that was first approved in 2002 in Japan under the trade name of Hachi-hachi. Analyses of TFP and its major metabolite, 4-[4-[(4-chloro-3-ethyl-1-methylpyrazol-5-yl)carbonylaminomethyl]phenoxy]benzoic acid (PTCA), in plasma obtained from a cadaver suspected to have died of TFP poisoning, were conducted by liquid chromatography-mass spectrometry. The existence of TFP and PTCA was confirmed by scan mode and quantitative analysis was performed by selected ion monitoring mode. Calibration curves showed good linearity over the range of 0.1-4 and 0.25-4 µg/mL, and concentrations were estimated to be 1.97 ± 0.02 and 2.88 ± 0.04 µg/mL for TFP and PTCA, respectively. The plasma extract was further examined to find other metabolites using quadrupole time-of-flight MS, and the results revealed three more metabolites, which were suggested to be hydroxy-TFP, dehydro-TFP and hydroxy-PTCA. Plausible metabolic pathways of TFP in humans are: (i) oxidation of the methyl group on the benzene ring, and (ii) hydroxylation followed by dehydration at the ethyl group on the pyrazole ring.

Multi mycotoxin analysis in food products using immunoaffinity extraction.

We developed a method for the simultaneous determination of deoxynivalenol, T-2 toxin, HT-2 toxin and zearalenone in wheat and biscuit by liquid chromatography/electrospray ionization/tandem mass spectrometry coupled with immunoaffinity extraction. This chapter describes a method to extract, clean-up, and quantitate these mycotoxins and the effect of the ion suppression of multifunctional column and IAC in the clean-up were compared.

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction.

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with immunoaffinity extraction is described. A clean-up was carried out using a DZT MS-PREP immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean-up were compared. The results with the DZT MS-PREP IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent-tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS-PREP IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03-0.33 ng x g(-1). From these studies, it is suggested that use of an IAC is effective in the clean-up of each mycotoxin, and, when combined with LC/ESI-MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity.

Determination of nivalenol and deoxynivalenol by liquid chromatography/atmospheric pressure photoionization mass spectrometry.

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2-100 ng x mL(-1) was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng x g(-1) and 0.39 ng x g(-1), respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels.

Time-of-flight mass spectrometry (TOF-MS) exact mass database for benzodiazepine screening.

Time-of-flight mass spectrometry coupled with liquid chromatography (TOF-MS) has been developed for screening and determination of benzodiazepines with an exact mass database. Benzodiazepines display similar chemical structures and molecular weights, and thus show similar mass spectra and protonated molecule ions. Discrimination of mass spectrometry at low resolving power using single liquid chromatography mass spectrometry (LC-MS) is commonly difficult. TOF-MS analysis was performed using a 1100 TOF (Agilent Technologies) equipped with a Zorbax C18 Extend column. Purine and fluorine compound solution was always introduced into the ion source, and real-time mass adjusting was performed. Specimens were prepared utilizing the liquid-liquid extraction procedure with 1-chlorobutane. Benzodiazepines are widely used in medical practice in Japan, and data acquired from TOF-MS measurements of 41 benzodiazepines, including active metabolites, were used to create an exact mass database. This database comprised molecular formulae, calculated exact masses and retention times. Calibrations were also included in a database. Precision for the 41 drugs was considered sufficient for quantitative analysis. In analysis of samples from patient who had taken > or =2 benzodiazepines, selectivity was improved using the TOF-MS exact mass database. TOF-MS is effective for forensic toxicology in discriminating between benzodiazepines with similar structure and metabolites.

Development of a liquid chromatography/time-of-flight mass spectrometric method for the simultaneous determination of trichothecenes, zearalenone and aflatoxins in foodstuffs.

A liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) method based on time-of-flight MS (TOFMS) with a real-time reference mass correction technique was developed for the simultaneous determination of Fusarium mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin, T-2 toxin, diacetoxyscirpenol, zearalenone) and Aspergillus mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2) in corn, wheat, cornflakes and biscuits. Samples were cleaned up with a MultiSep #226 column. Detection of the mycotoxins was carried out in exact mass chromatograms with a mass window of 0.03 Th. Calibration curves were linear from 2 to 200 ng x mL(-1) for trichothecenes and zearalenone, and 0.2 to 20 ng x mL(-1) for aflatoxins, by 20 microL injection. The limits of detection ranged from 0.1 to 6.1 ng x g(-1) in foodstuffs analyzed in this study. The LC/TOFMS method was found to be suitable for the screening of multiple mycotoxins in foodstuffs rapidly and with high sensitivity, and its performance was demonstrated for the confirmation for target mycotoxins.

[Mycological examination of domestic unpolished rice and mycotoxin production by isolated Penicillium islandicum].

Fungi growing on domestic rice were examined from April to June, 2003. One hundred samples of rice, which had been harvested in the autumn of 2002, were collected from the local market, and 15 samples of stored rice, which had been harvested in 2001 and stored in warehouses under government control, were used as samples. From each sample, 50 grains (100 grains in total) were plated on potato-dextrose agar (PDA) and malt yeast 40% sucrose agar (M40YA) containing chloramphenicol after being washed with sterile distilled water to remove any microorganisms on the surface, and incubated at 25 degrees C for a week. For most of the rice samples harvested in the preceding year, the proportion of grains infected with fungi was less than 20% of the total grains tested. In about half the samples of rice stored for one and half years, more than 80% of the grains were infected with fungi that grew on M40YA. The major genera of fungi isolated from the rice harvested in the preceding year were Penicillium and Alternaria, and those from the rice stored for one and a half years were Aspergillus, Penicillium and Eurotium. P. islandicum, A. versicolor, A. ochraceus and others were isolated as possible mycotoxin-producers in the mycoflora of domestic rice. P. islandicum was isolated from 3 samples, and 82% of the grains were infected with this fungus in one sample. All three isolates from these samples appeared to produce luteoskyrin on Czapek yeast extract agar, based on TLC and HPLC analysis.

Determination of carbamate pesticide residues in vegetables and fruits by liquid chromatography-atmospheric pressure photoionization-mass spectrometry and atmospheric pressure chemical ionization-mass spectrometry.

A liquid chromatography-atmospheric pressure photoionization (APPI)-mass spectrometry method was developed for the determination of 22 carbamates including their metabolites in vegetables and fruits. For the optimization of APPI, several APPI ion source parameters were examined. As a result, many carbamates with APPI using the optimized parameter gave simple mass spectra, and a strong signal corresponding to [M + H](+) was observed except for aldicarb. However, some carbamate metabolites gave ammonium adduct ions [M + NH(4)](+) as base peak ions. The mean recovery of each carbamate from grape and onion samples spiked at 5 ng/g was 81.7-105.7%, with relative standard deviations of 3.3-5.9%. Furthermore, matrix constituents did not significantly influence the ionization efficiency. The limit of detection (S/N = 3) in grape and onion was in the range of 0.33-3.33 ng/g. For the robustness of this method, this system has been used to analyze 50 samples, and the intensities for all carbamates were found to be unaffected by the contamination of the APPI source by sample matrix constituents. This result indicates that the method is reliable.

Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry.

A liquid chromatography-atmospheric pressure photoionization (APPI) mass spectrometry method was developed for the determination of chloramphenicol (CAP) in fish meats (young yellowtail and flatfish). For the optimization of APPI, several APPI ion source parameters and mobile phases were investigated. CAP with APPI using the optimized parameters gave simple mass spectra and a strong signal corresponding to [M-H]- was observed. Further, APPI was compared with atmospheric pressure chemical ionization (APCI) and APPI gave similar results in terms of structural information and a better signal-to-noise ratio. The samples were extracted with acetonitrile and evaporated to dryness followed by a clean-up step using the liquid-liquid distribution between acetonitrile and n-hexane. The mean recoveries of chloramphenicol from a young yellowtail meat and a flatfish meat spiked at 0.1-2 ng/g were 89.3-102.5 and 87.4-94.8%, respectively. The limit of detection (signal-to-noise ratio = 3) of the young yellowtail meat and the flatfish meat were 0.1 and 0.27 ng/g.

Liquid chromatography/mass spectrometric determination of patulin in apple juice using atmospheric pressure photoionization.

This paper describes a comparison between atmospheric pressure chemical ionization (APCI) and the recently introduced atmospheric pressure photoionization (APPI) technique for the liquid chromatography/mass spectrometric (LC/MS) determination of patulin in clear apple juice. A column switching technique for on-line extraction of clear apple juice was developed. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for both APCI and APPI. The results indicated that APPI provides lower chemical noise and signal suppression in comparison with APCI. The linear range for patulin in apple juice (correlation coefficient >0.999) was 0.2-100 ng mL(-1). Mean recoveries of patulin in three apple juices ranged from 94.5 to 103.2%, and the limit of detection (S/N = 3), repeatability and reproducibility were 1.03-1.50 ng mL(-1), 3.9-5.1% and 7.3-8.2%, respectively. The total analysis time was 10.0 min.

Determination of perfluorooctane sulfonate in river water by liquid chromatography/atmospheric pressure photoionization mass spectrometry by automated on-line extraction using turbulent flow chromatography.

A simple, fast and sensitive liquid chromatography/atmospheric pressure photoionization mass spectrometry (LC/APPI-MS) method, with automated on-line extraction using turbulent flow chromatography (TFC), was developed for the determination of perfluorooctane sulfonate (PFOS) in river water. In this method, following an on-line extraction by injection onto a column under TFC conditions, PFOS is back-flushed onto a reversed-phase column via on-line column switching, and resolved chromatographically at a laminar flow rate of 1 mL min(-1). Using this tandem LC-LC/APPI-MS system the extraction, separation and selective detection of PFOS in river water could be achieved with satisfactory selectivity and sensitivity. The limit of detection (LOD) (S/N = 3) and the limit of quantitation (LOQ) (S/N = 10)were 5.35 and 17.86 pg mL(-1). The described procedure was very simple since no off-line sample preparation was required, total analysis time being 18.75 min.