Biotransformation, trace analysis and effects of perfluoroalkyl and polyfluoroalkyl substances

Polyfluorierte und perfluorierte Verbindungen stellen eine Gruppe von Umweltkontaminanten dar, die aufgrund der starken C-F-Bindung besonders negative ökologische Eigenschaften besitzen. In dieser Arbeit wurde das Biotransformations-Potenzial von Fluortelomerethoxylaten (FTEO)unter aeroben Bedingungen untersucht. Nach Ermittlung der molekularen Zusammensetzung eines technischen FTEO-Gemischs mittels Elektrospray-Massenspektrometrie (ESI-MS) konnte aufgezeigt werden, dass zwei Transformationswege möglich sind, wobei einer dieser Wege über ω-oxidierte Carboxylate (FTEOCs), welche unter bestimmten Bedingungen nicht weiter abbaubar sind, verläuft. Ein anderer Abbauweg mündet in Perfluorcarboxylaten. Nach erfolgreicher Synthese zweier kurzkettiger FTEOCs konnten diese Verbindungen mithilfe einer Methode basierend auf Festphasenextraktion und HPLC-ESIMS/MS in einer Kläranlagenablaufprobe nachgewiesen und quantifiziert werden. Neben den FTEOCs konnten eine Reihe von Perfluorcarboxylaten und Perfluoralkansulfonaten in siebzehn Kläranlagenabläufen und vier Oberflächengewässern bestimmt werden. Die Untersuchung der Bioabbaubarkeit dreier potentieller Bausteine für neue, umweltfreundliche Fluortenside ergab, dass 6-(Trifluormethoxy)hexan-1-ol 100% anorganisches Fluorid freisetzte. Die homologe Verbindung 3(Trifluormethoxy)propan-1-ol hingegen setzte lediglich 15% Fluorid frei, wobei das verbleibende Fluor in der nicht weiter abbaubaren 3-(Trifluormethoxy)propansäure organisch gebunden blieb. Der dritte Baustein - 1-(2,2,3,3,4,4,4-Heptafluorbutoxy)propan-2-ol – führte erwartungsgemäß zur persistenten Verbindung Perfluorbutansäure. Die Toxizität der Perfluorbutansäure gegenüber der Algenspezies Pseudokirchneriella subcapitata und dem Kleinkrebs Chydorus sphaericus wurde anschließend ermittelt. Die toxikologischen Kenndaten ergaben keine akute Gefährdung dieser Organismen bei umweltrelevanten Konzentrationen. Weitere fluorierte Verbindungen wurden untersucht, wobei gezeigt werden konnte, dass die Toxizität mit zunehmender Kettenlänge im Allgemeinen zunimmt, die funktionelle Gruppe jedoch einen signifikanten Einfluss auf die Toxizität hat. Die massenspektrometrische Fragmentierung aller untersuchten Verbindungen wurde untersucht. Dabei ergab sich ein allgemeiner Fragmentierungsweg für Fluortelomer-basierte Verbindungen unter negativen Elektrospray-Bedingungen und stoßinduzierter Fragmentierung. Diese Erkenntnisse können in Zukunft zur Detektion neuer unbekannter Fluortelomerbasierter Verbindungen verwendet werden.Perfluoroalkyl and polyfluoroalkyl substances are environmental contaminants with adverse properties owing to the presence of the particularly strong C-F bond. In this thesis, the biotransformation potential of fluorotelomer ethoxylates (FTEOs) was assessed under aerobic conditions. After investigation of the technical FTEO mixture by electrospray-mass spectrometry (ESI-MS) techniques, it could be shown that two pathways are possible, one involving oxidation to ω-oxidized carboxylates (FTEOCs), which can be stable under certain conditions, and another pathway releasing perfluorocarboxylates, in function of the perfluoroalkyl chain length of the FTEO. After successful synthesis of two shortchain FTEOCs, these compounds were quantified in a wastewater treatment plant (WWTP) effluent by solid phase extraction and high performance liquid chromatography coupled to ESIMS/MS. Alongside the FTEOCs, a set of perfluorocarboxylates and perfluoroalkane sulfonates was determined in seventeen WWTP effluent samples and four surface water samples. The biotransformation behavior of three building blocks of potential novel environmentally friendly fluorosurfactants was investigated. It could be shown that 6-(trifluoromethoxy)hexan-1-ol released 100% inorganic fluoride, whereas 3-(trifluoromethoxy)propan-1-ol yielded only 15% fluoride. The remainder was shown to be organically bound as the non-degradable 3-trifluoromethoxy)propanoic acid. The building block 1-(2,2,3,3,4,4,4-heptafluorobutoxy)propan-2-ol expectedly released perfluorobutanoic as the persistent transformation product. The toxicity of the dead-end transformation product PFBA towards the green algae Pseudokirchneriella subcapitata and towards the Cladoceran Chydorus sphaericus was assessed with standardized tests. The results suggested no acute toxicity of these compounds at environmental levels. Other fluorinated compounds were included in the screening suggesting a general increase of toxicity with longer perfluoroalkyl chain length. However, the functional group itself showed a major effect on toxicity. The mass spectrometric fragmentation characteristics of the compounds studied in this thesis were carefully scrutinized. Comparison of different fluorotelomerbased compounds revealed a general fragmentation pattern for these compounds after negative electrospray ionization and collision-induced dissociation, which can be utilized to screen for novel fluorotelomer-based contaminants hereafter

High Resolution Mass Spectrometry of Polyfluorinated Polyether-Based Formulation

High resolution mass spectrometry (HRMS) was successfully applied to elucidate the structure of a polyfluorinated polyether (PFPE)-based formulation. The mass spectrum generated from direct injection into the MS was examined by identifying the different repeating units manually and with the aid of an instrument data processor. Highly accurate mass spectral data enabled the calculation of higher-order mass defects. The different plots of MW and the nth-order mass defects (up to n = 3) could aid in assessing the structure of the different repeating units and estimating their absolute and relative number per molecule. The three major repeating units were -C2H4O-, -C2F4O-, and -CF2O-. Tandem MS was used to identify the end groups that appeared to be phosphates, as well as the possible distribution of the repeating units. Reversed-phase HPLC separated of the polymer molecules on the basis of number of nonpolar repeating units. The elucidated structure resembles the structure in the published manufacturer technical data. This analytical approach to the characterization of a PFPE-based formulation can serve as a guide in analyzing not just other PFPE-based formulations but also other fluorinated and non-fluorinated polymers. The information from MS is essential in studying the physico-chemical properties of PFPEs and can help in assessing the risks they pose to the environment and to human health

Interspecies comparison of metabolism of two novel prototype PFAS

As a result of proposed global restrictions and regulations on current-use per-and polyfluoroalkyl substances (PFAS), research on possible alternatives is highly required. In this study, phase I in vitro metabolism of two novel prototype PFAS in human and rat was investigated. These prototype chemicals are intended to be safer-by-design and expected to mineralize completely, and thus be less persistent in the environment compared to the PFAS available on the market. Following incubation with rat liver S9 (RL-S9) fractions, two main metabolites per initial substance were produced, namely an alcohol and a short-chain carboxylic acid. While with human liver S9 (HL-S9) fractions, only the short-chain carboxylic acid was detected. Beyond these major metabolites, two and five additional metabolites were identified at very low levels by non-targeted screening for the ether- and thioether-linked prototype chemicals, respectively. Overall, complete mineralization during the in vitro hepatic metabolism of these novel PFAS by HL-S9 and RL-S9 fractions was not observed. The reaction kinetics of the surfactants was determined by using the metabolite formation, rather than the substrate depletion approach. With rat liver enzymes, the formation rates of primary metabolite alcohols were at least two orders of magnitude higher than those of secondary metabolite carboxylic acids. When incubating with human liver enzymes, the formation rates of single metabolite carboxylic acids, were similar or smaller than those experienced in rat. It also indicates that the overall metabolic rate and clearance of surfactants are significantly higher in rat liver than in human liver. The maximum formation rate of the thioether congener exceeded 10-fold that of the ether in humans but were similar in rats. Overall, the results suggest that metabolism of the prototype chemicals followed a similar trend to those reported in studies of fluorotelomer alcohols.</p

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.This project was financially supported by the Deutsche José Carreras Leukämie Stiftung grant DJCLS 20R/2017 (to L.V., S.H., L.M.S., and A.T.), the Emerson foundation grant 643577 (to L.V. and L.M.S.) and the German Bundesministerium für Bildung und Forschung (BMBF) through the Juniorverbund in der Systemmedizin “LeukoSyStem” (FKZ 01ZX1911D to L.V., S.H., and S.R). Contributions by S.R. were further supported by Emmy Noether Fellowship RA 3166/1-1 (DFG). Contributions by C.P. were supported by a Max-Eder Grant (German Cancer Aid 70111531). Contributions by D.N., J.C.J., W.K.H., and T.B. were supported by the Gutermuth Foundation, the H.W. & J. Hector fund, Baden-Württemberg, and the Dr. Rolf M. Schwiete Fund, Mannheim. D.N. is an endowed professor of the Deutsche José Carreras Leukämie Stiftung (DJCLS H 03/01