We first fractionated the metabolites and analyzed the organic phase profile obtained with 25°C fungi cultures, to obtain enough metabolites to be seen in HPLC analysis. For 25°C fungi cultures, we detected and identified fumigaclavine A and C, fumitremorgin C, verruculogen, and fumagillin by comparison with authentic standards (Figure 1). For 37°C culture, other metabolites were identified on the basis of their UV spectrum: fumigaclavine A, gliotoxin, fumiquinazoline F, fumitremorgin C, fumagillin, and helvolic acid. When testing the different HPLC fractions and commercial products, the results obtained after 30 min or 3 hours of exposure were similar. Therefore, only the 30 min results were retained.
At 37°C, only the 20–30 min fraction 3 (F 3) increased the Vt (Figure 2). As this fraction was known to contain the secondary metabolites helvolic acid, fumagillin and verruculogen, the effects of different concentrations of these commercially available compounds were tested. Helvolic acid had no significant effect (data not shown). The effects of fumagillin are summarized in Figure 3 and were opposite to the effects of F3. Only verruculogen showed effects similar to those of F3. In Figure 4, the effects of different concentrations of verruculogen are presented, ranging from 10-4 to 10-9 M. The effects on Rt and Vt were slightly dissociated, as 10-4 and 10-6 M verruculogen decreased Rt and increased Vt, whereas 10-7 and 10-8 M verruculogen only increased Vt with no significant effect on Rt.
To verify that verruculogen was responsible for the effects of fraction 3, F3 was further fractionated into five two-minute fractions (F'1-5). In these HPLC conditions, verruculogen was recovered in F'3 (24–26 min) while helvolic acid and fumagillin were eluted together in F'5 (28–30 min). Among the five tested fractions, only F'3 displayed results similar to those of F3. Since verruculogen is produced in greater amounts at 25°C than at 37°C [15], we performed similar experiments with the organic phase obtained at 25°C to look for a concentration effect. For 25°C culture, F'3 revealed an increase in Vt and a decrease in Rt within the same range as 10-6 M standard verruculogen, whereas the effects observed with F'3 of 37°C culture were similar to those obtained with lower concentrations of verruculogen (data not shown).
Fraction F'3 was analyzed by mass spectrometry and verruculogen was identified on the basis of electron ionization MS and UV spectral analysis, after comparison with a reference standard. In the analysis of fraction F'3, the mass spectrum of verruculogen showed two major ions at m/z 494 and 534, corresponding to loss of water and sodium adduct, respectively (Figure 5). The collision-induced dissociation MS-MS spectrum of the 494 ion led to fragment at m/z 410, identical to that of authentic verruculogen [16].
The amounts of fumagillin and verruculogen were estimated by HPLC-DAD using standard curves. F'3 contained 4.15 μg of fumagillin for 37°C culture and 3.96 μg for 25°C culture. Therefore, the concentration of fumagillin in culture filtrate used to challenge the HNEC was 0.36 10-6 M, more than 300 times lower than the lowest concentration, 1.1 10-4 M, of purified fumagillin resulting in a significant effect (Figure 3), thus definitively excluding fumagillin as the toxin involved in our observations. For verruculogen, F'3 of 37°C culture contained less than 80 ng of verruculogen and Fraction F'3 of 25°C culture contained 2.1 μg of verruculogen. The concentrations obtained after resolubilization in DMSO and dilution to challenge HNEC corresponded to 0.6 10-6 M for 25°C culture and 1.4 10-8 M of verruculogen for 37°C culture. Compared with the results obtained with the serial dilutions of standard verruculogen (Figure 4), fraction F'3 of 25°C culture showed an increase in Vt and Rt, as did the 10-6 M concentration, whereas F'3 of 37°C culture presented a decrease in Vt in the same way as the 10-8 concentration.
Verruculogen was detected by HPLC-DAD in the 67 A. fumigatus hyphal and conidial extracts tested, from 0.13 to 17.2 μg/g of wheat. One extract of conidial fractions was also analyzed by mass spectrometry to verify the presence of verruculogen. In positive mode, verruculogen (molecular weight: 511 g/mol) was detected in conidial extract. The presence of verruculogen was confirmed after 14.8 min of elution, at which time its mass spectrum corresponded to that of standard verruculogen. In these extracts, seven other compounds were also specifically detected (emodin, gliotoxin, fumigaclavine C and four compounds of the tremorgen family, namely tryprostatin A, TR2, fumitremorgin C, and dihydroxy-fumitremorgin C).
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