# Effects of Workers Exposure to Nanoparticles Studied by NMR Metabolomics
## 4 Conclusions  
The EBC and blood plasma samples of a cohort of 20 workers exposed to NPs during their occupation were analysed by \({}^{1}\)H NMR spectroscopy and processed by statistical analysis. Altogether, 15 metabolites were identified in EBC samples, while the analysis of plasma samples provided 58 metabolites. Subsequent multivariate statistical analyses performed on binning data from EBC and concentrations of 58 metabolites from plasma samples enabled clear discrimination between the pre-shift, post-shift and control groups. The univariate statistical analysis revealed statistically significant metabolites. Although plasma and EBC samples each showed changes in levels of different metabolites, the metabolic pathway analysis indicated, in both cases, mainly a reaction of the organism to oxidative stress and subsequent efforts for its protection. The comparison of the pre-shift and post-shift group accompanied by comparison of the post-shift and control group provided insight into the acute effect of the NPs exposure. Altered levels of lactate, pyruvate, 3-hydroxybutyrate, mannose and \(O\)-acetylcarnitine indicated an energy balance impairment. The altered levels of glutamate, cystine, tryptophan, acetate, propionate and butyrate were associated to the pathways related to the production of antioxidants, mainly glutathione, and other protective species. The comparison of the pre-shift and control group revealed that the chronic effect of the NPs exposure manifested mainly in an alteration in glutamine and glutamate metabolism. The increased levels of hypoxanthine indicated an impairment of the purine metabolism pathway. The presented results correspond well with similar studies performed on cohorts exposed to different types of NPs, indicating that the observed adverse effects can be attributed to nanoparticles in general, rather than to their chemical nature. This work is one of the few dealing with the occupational exposure to NPs studied by the means of NMR metabolomics. Similar response to NPs exposure was observed for both types of samples indicating that either biofluid can be used for evaluation of adverse effects of nanoparticles inhalation. Potentially, blood derivatives could serve as an alternative to commonly used EBC samples. **Supplementary Materials:** The following are available online at [[https://www.mdpi.com/article/10.3390/app11146601/s1](https://www.mdpi.com/article/10.3390/app11146601/s1)]([https://www.mdpi.com/article/10.3390/app11146601/s1](https://www.mdpi.com/article/10.3390/app11146601/s1)), Table S1: Basic characteristics of the samples; Figure S1: 1H NMR spectrum of a representative BBC sample; Figure S2: 1H NMR spectrum of a representative blood plasma sample; Table S2: Wilcoxon test for EBC samples; Figure S3: Principal component analysis for EBC samples; Figure S4: Principal component analysis for plasma samples; Table S3: Wilcoxon test for blood plasma samples; Figure S5: Fold change projection of pre-shift subjects and healthy controls; Figure S6: Fold change projection of post-shift subjects and healthy controls; Figure S7: Fold change projection of pre-shift and post-shift subjects; Figure S8: Metabolic pathway analysis; Table S4: Overview of the most influenced metabolic pathways; Table S5: Wilcoxon test for EBC samples after exclusion of smoking subjects; Table S6: Wilcoxon test for blood plasma samples after exclusion of smoking subjects; Figures S9 and S10: Multivariate statistical analysis of EBC samples after exclusion of smoking subjects; Figures S11 and S12: Multivariate statistical analysis of plasma samples after exclusion of smoking subjects. **Author Contributions:** Conceptualization, V.Z., S.D. and D.P. ; methodology, S.H., L.M. and J.S. ; formal analysis, J.S. and D.P. ; investigation, S.H. and L.M. ; resources, J.S., V.Z. and D.P. ; data curation, S.H., L.M., J.S. and S.V. ; writing--original draft preparation, S.H. and L.M. ; writing--review and editing, J.S. ; visualization, S.H., L.M. and J.S. ; supervision, J.S. ; project administration, S.V. and D.P.