Acute toxicity testing of the tire rubber-derived chemical 6PPD-quinone on Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

Recent identification of 6PPD-quinone as the chemical causing acute toxicity in coho salmon has led to substantial concern regarding toxicity of this contaminant for other aquatic species. Environmental occurrence of 6PPD-quinone is probably high as it is an oxidation product of a common tire rubber additive. Research on 6PPD-quinone toxicity in fish has revealed a rather unusual pattern, with closely related species exhibiting responses ranging from extreme sensitivity to no effect. Of eleven previously studied fish species, 6PPD-quinone was toxic to four. The species-specific toxicity of 6PPD-quinone complicates urgently needed environmental risk assessment. We investigated acute toxicity of 6PPD-quinone in Atlantic salmon and brown trout alevins (sac fry). These species have previously not been tested for sensitivity to 6PPD-quinone. The fish were exposed in static conditions in eight treatments with initial concentrations ranging from 0.095–12.16 µg/L. Fish were observed for 48 h and changes in concentrations of 6PPD-quinone were monitored throughout the experiment. No mortalities or substantial changes in behaviour were recorded in either Atlantic salmon or brown trout. This provides an important first step in assessing effects of 6PPD-quinone on these highly economically and culturally important species.acceptedVersio

Presence of 6PPD-quinone in runoff water samples from Norway using a new LC–MS/MS method

The chemical 6PPD-quinone is highly toxic to some fish species of the Oncorhynchus and Salvelinus genera and is the oxidation product of the common car tire additive 6PPD. We present a new sample preparation method that involves liquid-liquid extraction of water samples followed by silica-based solid phase extraction prior to LC–MS/MS analysis. The new sample preparation method showed good analyte recovery from spiked water samples (78%–91%) and a low ion suppression effect, surpassing previously published methods. This new method was successfully validated, achieving a limit of quantification of 5 ng/L and estimated expanded measurement uncertainty of 18.6%. In a proof-of-concept study, the method was applied to several water samples from various sources in Southern Norway. These were runoff samples from tunnel washing, from a tunnel runoff treatment plant and downstream of the plant drain. In addition, two water samples from puddles were included: one was run-off from an artificial soccer turf field and one from a puddle on a country road. The results of the analyses revealed that the concentration of 6PPD-quinone was above the LC50 reported for coho salmon (Oncorhynchus kisutch) in all samples except the samples from and downstream of the treatment plant. The highest measured concentration was 258 ng/L, which is the 2.7-fold of the reported LC50 in coho salmon (95 ng/L). Our initial data emphasize the need for more comprehensive environmental monitoring of 6PPD-quinone as well as toxicological studies in aquatic organisms. car tire, liquid chromatography, mass spectrometry, runoff water, artificial turf pitch, 6PPDQ, 6PPD-quinonepublishedVersio

Identification of Caribbean ciguatoxins from benthic dinoflagellates advances knowledge on toxin chemistry and analysis

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N-terminal acetylation levels are maintained during acetyl-CoA deficiency in Saccharomyces cerevisiae

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality control and stress tolerance. Despite its prevalence, the mechanisms regulating Nt-acetylation are still nebulous. Here, we present the first global study of Nt-acetylation in yeast cells as they progress to stationary phase in response to nutrient starvation. Surprisingly, we found that yeast cells maintain their global Nt-acetylation levels upon nutrient depletion, despite a marked decrease in acetyl-CoA levels. We further observed two distinct sets of protein N termini that display differential and opposing Nt-acetylation behavior upon nutrient starvation, indicating a dynamic process. The first protein cluster was enriched for annotated N termini showing increased Nt-acetylation in stationary phase compared with exponential growth phase. The second protein cluster was conversely enriched for alternative nonannotated N termini (i.e. N termini indicative of shorter N-terminal proteoforms) and, like histones, showed reduced acetylation levels in stationary phase when acetyl-CoA levels were low. Notably, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the steady-state levels of these proteins were regulated both by starvation and NatA activity. In summary, this study represents the first comprehensive analysis of metabolic regulation of Nt-acetylation and reveals that specific, rather than global, Nt-acetylation events are subject to metabolic regulation

Presence of 6PPD-quinone in runoff water samples

Dataset of Agilent raw files related to upcomming publication </ul

Fast and Quantitative Phospholipidomic Analysis of SH-SY5Y Neuroblastoma Cell Cultures Using LC-MS/MS and 31P NMR

Global lipid analysis still lags behind proteomics with respect to the availability of databases, experimental protocols, and specialized software. Determining the lipidome of cellular model systems in common use is of particular importance, especially when research questions involve lipids directly. In Parkinson’s disease research, there is a growing awareness for the role of the biological membrane, where individual lipids may contribute to provoking α-synuclein oligomerisation and fibrillation. We present an analysis of the whole cell and plasma membrane lipid isolates of a neuroblastoma cell line, SH-SY5Y, a commonly used model system for research on this and other neurodegenerative diseases. We have used two complementary lipidomics methods. The relative quantities of PC, PE, SMs, CL, PI, PG, and PS were determined by 31P NMR. Fatty acid chain composition and their relative abundances within each phospholipid group were evaluated by liquid chromatography–tandem mass spectrometry. For this part of the analysis, we have developed and made available a set of Matlab scripts, LipMat. Our approach allowed us to observe several deviations of lipid abundances when compared to published reports regarding phospholipid analysis of cell cultures or brain matter. The most striking was the high abundance of PC (54.7 ± 1.9%) and low abundance of PE (17.8 ± 4.8%) and SMs (2.7 ± 1.2%). In addition, the observed abundance of PS was smaller than expected (4.7 ± 2.7%), similar to the observed abundance of PG (4.5 ± 1.8%). The observed fatty acid chain distribution was similar to the whole brain content with some notable differences: a higher abundance of 16:1 PC FA (17.4 ± 3.4% in PC whole cell content), lower abundance of 22:6 PE FA (15.9 ± 2.2% in plasma membrane fraction), and a complete lack of 22:6 PS FA.publishedVersio

Identification and cross-species comparison of in vitro phase I brevetoxin (BTX-2) metabolites in northern Gulf of Mexico fish and human liver microsomes by UHPLC-HRMS(/MS)

Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72–99% reduction within 1 h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs

Reductive Amination for LC–MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes

Deconvolution of Mixture Spectra and Increased Throughput of Peptide Identification by Utilization of Intensified Complementary Ions Formed in Tandem Mass Spectrometry

A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high-resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial data set