Biochemical markers for the detection and classification of Aspergillus

The genus Aspergillus includes a diverse group of filamentous fungi that are widely distributed in nature, commonly found in soil. The Aspergilli include species that can be beneficial or detrimental to humans, so detection and accurate identification of these organisms can be very important. Morphology and genetic sequence analysis are well established methods for classifying and identifying fungi, but morphology remains a widely used technique that generally works well for Aspergilli. However, some organisms may be misidentified due to atypical morphology and some hidden (cryptic) species may not be recognized as different from named species based on readily observable traits. In this study, reference strains of different Aspergillus species, Penicillium chrysogenum, Candida albicans, and Cryptococcus neoformans were characterized using LC/MS and GC/MS biochemical profiling techniques in order to find specific small molecules, peptides or biochemical profiles that can be used in addition to established methods to detect and classify Aspergilli to the species level. Subsequently, analytical methods developed for characterizing the reference strains were applied, along with morphology and PCR, to characterize and identify several laboratory and field isolates. Some unique compounds and biochemical patterns did emerge from small molecule profiling that could be used for classifying Aspergilli, but protein profiling by LC/MS/MS was a much more effective approach. Tandem mass spectra from LC/MS/MS of tryptic peptides from fungal proteins were searched against protein databases and matched to theoretical spectra derived from those databases. Many of the amino acid sequences detected were taxonomically diagnostic for classifying Aspergillus species. Protein profiling also provided a great deal of additional biochemical information on the test organisms by identifying the predominant enzymes and structural proteins present under different experimental conditions and may find broader application for identifying and studying other organisms

Quantification of urinary aflatoxin B1 dialdehyde metabolites formed by aflatoxin aldehyde reductase using isotope dilution tandem mass spectrometry

The aflatoxin B1 aldehyde reductases (AFARs), inducible members of the aldo-keto reductase superfamily, convert aflatoxin B1 dialdehyde derived from the exo- and endo-8,9-epoxides into a number of reduced alcohol products that might be less capable of forming covalent adducts with proteins. An isotope dilution tandem mass spectrometry method for quantification of the metabolites, C-8 monoalcohol, dialcohol, and C-6a monoalcohol, was developed to ascertain their possible role as urinary biomarkers for application to chemoprevention investigations. This method uses a novel 13C 17-aflatoxin B1 dialcohol internal standard, synthesized from 13C17-aflatoxin B1 biologically produced by Aspergillus flavus. Chromatographic standards of the alcohols were generated through sodium borohydride reduction of the aflatoxin B1 dialdehyde. This method was then explored for sensitivity and specificity in urine samples of aflatoxin B1-dosed rats that were pretreated with 3H-1,2-dithiole-3-thione to induce the expression of AKR7A1, a rat isoform of AFAR. One of the two known monoalcohols and the dialcohol metabolite were detected in all urine samples. The concentrations were 203.5 ± 39.0 ng of monoalcohol C-6a/mg of urinary creatinine and 10.0 ± 1.0 ng of dialcohol/mg of creatinine (mean ± standard error). These levels represented about 8.0 and 0.4% of the administered aflatoxin B1 dose that was found in the urine at 24 h, respectively. Thus, this highly sensitive and specific isotope dilution method is applicable to in vivo quantification of urinary alcohol products produced by AFAR. Heretofore, the metabolic fate of the 8,9-epoxides that are critical for aflatoxin toxicities has been measured by biomarkers of lysine-albumin adducts, hepatic and urinary DNA adducts, and urinary mercapturic acids. This urinary detection of the alcohol products directly contributes to the goal of mass balancing the fate of the bioreactive 8,9-epoxides of AFB1 in vivo. © 2008 American Chemical Society