Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.

Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism Caenorhabditis elegans to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) parg-1 or parg-2 (two orthologue of PARG), indicates that parg-1 accounts for most of the glycohydrolase activity in worms.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics.

BACKGROUND: Lipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. RESULTS: A SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SIGNIFICANCE: This method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.