My research focuses on the development of a novel method for the fast detection of aflatoxin-producing fungi from the volatile organic compounds that they produce. Aflatoxins have received great attention because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. Traditional detection and quantification techniques are considered time-consuming, high cost, and require technical professionals. The `odor' or so called volatile metabolites released by a fungus is the key for fast detection. Several researchers have reported that diverse fungi species have unique volatile metabolite patterns. This study focuses on answering several questions: Is it possible to discriminate aflatoxins-producing fungi from other fungi based on volatile metabolites? What are the key discriminating biomarkers related to each fungus? Does the growth environment have an effect on the production of volatile metabolites? What chemicals are consistently emitted by a fungus under varied conditions? To answer these questions, one toxigenic and one nontoxigenic A. flavus isolate were studied to evaluate the microbial volatile organic compound (MVOC) profiles. The results described in chapter two of this dissertation indicate that MVOC production is time-dependent and that aflatoxigenic and non-aflatoxigenic strains have different MVOC expression patterns. Chapter three describes the effects of experimental parameters on fungal volatile metabolites. The identity and quantity of MVOCs can be affected by many factors including SPME fiber type, fungal growth media, and growth temperature. A CAR/PDMS coated fiber performed better than the other SPME fibers by collecting a larger variety and quantity of MVOCs. Fungi grown on the chemical defined liquid media produced much larger quantities of MVOCs compared to the other media. The highest MVOC production results were found at 30 degrees Celsius. The fungi discrimination study was extended in chapter four by including 3 toxigenic and 3 non-toxigenic isolates using multivariate analysis. The results indicate that volatile patterns vary even at the fungal isolate level and that discrimination of aflatoxin-producing fungi from non-toxigenic fungi is possible.

Metabolomics is an emerging field that entails the detailed characterization of the ensemble of metabolites produced by living organisms; subfields include drug metabolism and natural environmental toxin production. The first part of the dissertation pursued metabolism of glyceollins, i.e., isoflavones produced by soybeans, that are potential cancer therapy agents. In vivo glyceollin metabolites produced in rats were investigated by on-line Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry. An odd-electron fragment ion at m/z 148, formed in violation of the even-electron rule, and diagnostic of the glyceollin backbone, was discovered. Based on this finding, a negative mode precursor ion scanning method was developed to screen for glyceollins and their metabolites from biological samples. Products of both Phase I and Phase II metabolism were identified, none of which have been previously reported. Sulfated metabolites were confirmed by accurate mass measurement, while glucuronide conjugation was confirmed by enzyme-assisted glucuronidation by rat liver microsomes. Intact GSH-glyceollin conjugates were not observed, but breakdown products of the GSH pathway, i.e., cysteinylglyceine, cysteine, and acetylated cysteine, were identified as conjugates of oxygenated glyceollins. The identification of GSH by-product conjugates was confirmed in product ion spectra acquired in the negative mode (where peptide anions, and glyceollin-bearing cleaved peptide portions were observed), as well as in the positive mode (where intact oxygenated glyceollin fragments appeared without the initially-present peptide portion). Mass spectral evidence strongly supports a metabolic pathway involving initial epoxidation of glyceollins followed by GSH addition at the epoxidation site. The second part of the dissertation undertook the investigation of secondary metabolites called microbial volatile organic compounds (MVOCs) produced by fungi (mold) that have been reported to have adverse human health effects. MVOCs were collected onto different sorbent materials and analyzed by Thermal Desorption Analysis coupled with on-line Gas Chromatography-Mass Spectrometry. Fungal MVOCs were characterized from various simulated flooding conditions (brackish, freshwater, and saltwater) and different substrates (nutrient rich vs. low nutrient) to determine diagnostic MVOCs. Ten fungi from simulated environments were identified by genetic sequencing. Cladosporium sp. and Chaetomium sp. were cultivated and their emitted MVOCs, 3-furaldehyde and 3-(4-hydroxy-3-methoxyphenyl)-2-propenal, were proposed as diagnostic indicators of these fungi.

Metabolomics is a powerful science that can be applied for the discovery of disease biomarkers, and investigation of altered metabolomes due to abiotic and biotic perturbations. This dissertation is focused on untargeted metabolomic applications to investigate fungal metabolite alterations associated with pathogenicity, fungal disease propagations, and symbiosis. This dissertation employs qualitative analysis of metabolite mixtures using HS-SPME coupled GC-MS and TMS derivatization followed by GC-MS analytical platforms. In the first study, we discovered a biomarker combination to diagnose fungal soft tissue disease in sweet potato at an early stage of disease propagation. We used an HS-SPME GC-MS untargeted metabolomics workflow to analyze the VOC associated with Rhizopus stolonifer infected and healthy sweet potatoes in situ and simulated warehouse environments. A single combination of 4 biomarkers was able to diagnose R. stolonifer fungal soft tissue disease (AUC = 0.980, 95% C.I. 0.937-1) and the early stage of the fungal soft rot disease (AUC = 0.999, 95% C.I. 0.978-1). We were able to detect the biomarkers: 1- propanol, ethyl alcohol, ethyl propionate and 3-methyl-3- buten-1-ol during disease progression in a simulated warehouse environment. Therefore, this study shows the feasibility of early diagnosis of fungal soft tissue disease by a real-time screening of volatile profiles of sweet potato in post-harvest storage. When considering the study of a particular species metabolome, it is crucial to develop a metabolite extraction protocol. In the second study, the performance of the six different metabolite extraction solvents mixtures was tested with the preferred mix being: butanol:methanol:water (2:1:1, v/v at -20 °C) which was used as a single solvent mix to extract both polar and relatively non-polar metabolites simultaneously in a single extraction step. The Macrophomina phaseolina fungal metabolome was investigated using the solvent mix. Finally, fungal mutualism was studied using untargeted metabolomics. Most often mycorrhizal metabolomics workflows are based on analyzing the Arbuscular Mycorrhizae colonized root metabolome. But here, we used hyphal materials to examine the mutualistic symbiotic association of the AM fungi. All untargeted metabolomic studies included chemometric data analysis and specific biomarkers and or metabolites were determined using multivariate statistics or prediction model building and validating.

People spend most of their time indoors and mostly in the dwelling. It is therefore important to investigate associations between indoor exposure in dwellings and health. Symptoms that may be related to the indoor environment are sometimes referred to as the "sick building syndrome" (SBS). SBS involves symptoms such as eye, skin and upper airway irritation, headache and fatigue. Three longitudinal studies and one prevalence study on personal and environmental risk factors for SBS in adults were performed. The prevalence study included measurements of indoor exposures in the dwellings. The longitudinal studies, with 8-10 years follow-up time, showed that smoking and indoor paint emissions were risk factors for SBS. Moreover, building dampness and moulds in dwellings were risk factors for onset (incidence) of general symptoms, skin symptoms and mucosal symptoms. In addition subjects living in damp dwellings have a lower remission of general symptoms and skin symptoms. Hay fever was a risk factor for onset of skin symptoms and mucosal symptoms, and asthma was a risk factor for onset of general and mucosal symptoms. Biomarkers of allergy and inflammation (bronchial reactivity, total IgE, ECP and eosinophil count) were predictors of onset of SBS symptoms, in particular mucosal symptoms. In the prevalence study, any SBS-symptom was associated with some individual volatile organic compounds of possible microbial origin (MVOC) e.g. 2-pentanol, 2-hexanon, 2-pentylfuran and 1-octen-3ol. Moreover, there were associations between indoor levels of formaldehyde and the plasticizer Texanol and any SBS. The result from the study indicates that individual MVOC are better indicators of SBS than the total value of MVOC. A final conclusion is that smoking, dampness and moulds and emissions from indoor painting may increase the onset of SBS. The indoor environment in dwellings over time has improved, but there is still a need for further improvements of the indoor environment in dwellings. More longitudinal SBS studies are needed.

Microorganisms are able to produce a wide variety of volatile organic compounds. This thesis deals with sampling, analysis and identification of such compounds, produced by microorganisms commonly found in buildings. The volatiles were sampled on adsorbents and analysed by thermal desorption cold trap-injection gas chromatography, with flame ionization and mass-spectrometric detection. The injection was optimized, with respect to the recovery of adsorbed components and the efficiency of the chromatographic separation, using multivariate methods. Eight adsorbents were evaluated with the object of finding the most suitable for sampling microbial volatiles. Among the adsorbents tested, Tenax TA proved to have the best properties for the purpose. Some carbon-containing adsorbents, e.g., Tenax GR and Carbopack B, showed a catalytic effect on thermal decomposition of some compounds, mainly terpene derivatives. Five fungal species, Aspergillus versicolor, Pénicillium commune, Cladosporium cladosporioides, Paecilomyces variotii and Phialophora fastigiata, and anactinomycete, Streptomyces albidoflavus, were cultivated on various artificial media and/or building materials. Cultivation was performed in culture flasks, provided with air inlet and outlet tubes. Humidified air was constantly led through the flasks, and samples were taken by attaching adsorbent tubes to the outlet tubes of the flasks. The cultivation medium proved to be of vital importance for metabolite production, quantitatively as well as qualitatively. For Streptomyces albidoflavus the effect of medium, cultivation temperature, and oxygen and carbon dioxide levels in the supplied air on the production of volatiles, was studied using multivariate techniques. The medium and the temperature exerted the largest influence, but the oxygen and carbon dioxide levels also affected the amounts of some metabolites produced. The produced volatile metabolites were identified by mass spectrometry and reference compounds. Alcohols, ketones, sulphur compounds and terpenes were most frequently found, but hydrocarbons, ethers and esters were also produced by some species. Among the most commonly produced metabolites, which are also suggested as potential indicator substances for excessive growth of microorganisms in buildings, were 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanone, 3-methyl-2-pentanone, dimethyl disulphide, -methylfuran, 2,5-dimethylfuran and geosmin. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu

Résumé: De nombreuses études ont établi que l’exposition aux moisissures intérieures peut être nuisible à la santé. Dans une récente étude, 21 composés organiques volatils microbiens (COVM) ont été sélectionnés comme biomarqueurs potentiels de l’exposition aux moisissures intérieures. L’objectif du projet était de déterminer les coefficients de partage (CP) sang:air, urine:air, plasma:air et eau:air pour ces composés, paramètres utilisés dans la prédiction de la toxicocinétique des xénobiotiques. Pour y parvenir, la méthode de flacons à l’équilibre a été utilisée. Les COVM (1.57-2.01 µg) injectés dans des flacons (20 ml) hermétiquement scellés contenant soit 0.5 ml de matrice (flacons test) ou simplement de l’air (flacons références) ont été incubés 60 min à 37⁰ C sous agitation continue. La quantification des COVM dans l’espace de tête des flacons (test et références) a été ensuite faite par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS/MS). Les CP de 19 COVM variaient entre 78 et 4721 pour sang:air, 14 et 3586 pour urine:air, 64 et 5604 pour plasma:air et 16 et 2210 pour eau:air. Les CP eau:air étaient étroitement liés aux CP urine:air pour 17 COVM (R2 = 0,97, pente =1,001) suggérant que la valeur de CP eau:air (lorsqu’inférieur à103) peut être un substitut au CP urine:air. L’étude des ratios sang:urine indique que six COVM seraient nettement plus concentrés dans le sang, matrice de choix pour ces composés. Les données générées par cette étude faciliteront le développement de modèles pharmacocinétiques de COVM et le paramétrage de leurs prélèvements en tant que biomarqueurs de l’exposition aux moisissures intérieures. / Abstract: Numerous studies have established that exposure to indoor molds can be harmful to health. In a recent study, 21 microbial volatile organic compounds (mVOCs) were selected as potential biomarkers of indoor mold exposure. The objective of the project was to determine the blood:air, urine:air, plasma:air, and water:air partition coefficients (PCs) for these compounds, parameters used in predicting the toxicokinetics of xenobiotics. To achieve this, the vial-equilibration method was used. mVOCs (1.57-2.01 µg) were injected into hermetically sealed vials (20 ml) containing either 0.5 ml of matrix (test vials) or simply air (reference vials) and were incubated for 60 min at 37 ⁰C under continuous agitation. Quantification of mVOCs in the headspace of the vials (test and reference) was then performed by gas chromatography mass spectrometry (GC-MS/MS). The PCs of 19 mVOCs ranged from 78 and 4721 for blood:air, 14 and 3586 for urine:air, 64 and 5604 for plasma:air, and 16 and 2210 for water:air. Water:air PCs were closely related to urine:air PCs for 17 mVOCs (R2 = 0.97, slope =1.001) suggesting that the water:air PC value (when less than 103) may be a surrogate for the urine:air PC. The blood:urine ratio study indicates that six mVOCs would be significantly more concentrated in blood, the matrix of choice for these compounds. The data generated from this study will facilitate the development of pharmacokinetic models of mVOCs and the parameterization of their sampling as biomarkers of indoor mold exposure.