Further insights into carbon source usage in the

Further insights into carbon source usage in the presence of the xenobiotic were achieved by focusing on the sugar metabolism. Therefore, this study primarily focused on AM 114 (glycolysis, TCA cycle) or glucose metabolism-related compounds and included 32 compounds analyzed in the MRM mode with a microLC–MS/MS instrument. The applied LC–MS/MS method and sample preparation procedure were developed based on a multi-method developed by Wei et al. [27] and modified for mycelia material extraction and analysis. The LC–MS/MS data from the control(c) and alachlor-containing (ala) cultures were subjected to principal component analysis (PCA) [33] with the MarkerView software (AB Sciex, USA). The major differences occurred during the first 24 h of culture, as presented in Fig. 5, where the samples ‘0h’ and ‘ala24’ are located farthest from each other and from the other samples on the PCA chart. The location of the samples on the chart is most affected by high amounts of methylmalonate/succinate and UDP-glucose (samples ‘ala24’), malate and aconitate (samples ‘0h’) and citrate (the rest of the samples) (Fig. 5). Notable variation was also observed within the other samples, and the sample groups ‘c24’, ‘ala72’, ‘ala 120 and ala168’ and ‘c120 and c168’ tend to form clusters in different locations compared with each other. To examine all of the data, the PCA loadings for each analyte (peak areas) were averaged and recalculated as percentage values (such that 100% is the highest loading for the analyte). The data are presented in Table 1, and a heat map and simple chart scoring were applied to facilitate data evaluation. The data revealed significant increases in the relative concentration to the maximum or a high level of the selected compounds, particularly hexose mono- and diphosphates, glycerol/glycerate group and end products of the TCA cycle (succinate, fumarate, malate, oxaloacetate), after 24 h of culture in the alachlor-containing cultures. Similar behavior was observed for acetyl-CoA. However, the citrate concentration was lower after 24 h in the alachlor-containing cultures in comparison to the control samples. In contrast, the citrate concentrations in both cultures were similar at all other tested time points. The maximum relative concentration in the ‘ala24’ samples was also observed for UDP-glucose/galactose, which is significant because in all of the other samples, the relative concentration was 70–90% lower. Interestingly, the ascorbate and 3-phosphoglycerate concentrations were significantly increased in the ‘ala24’, ‘ala72’ and ‘ala120’ samples in comparison to the other samples.