In-plate toxicometabolomics of single zebrafish embryos

Abstract

A high-throughput toxicometabolomic assay involving single zebrafish embryos was developed which links apical endpoints to biochemical perturbations.

Microbiota-dependent and independent production of L-dopa in the gut of Daphnia magna

“…Instrumental analysis was carried out through injection on the HPLC-HRMS system (Ultimate 3000, ThermoFisher Scientific; Q Exactive HF Orbitrap, ThermoFisher Scientific) with an adapted version of the electrospray ionization settings used in Ribbenstedt et al [24] (positive ionization, 3700V, sheath gas 30, aux gas 10, sweep gas 0, S-lens RF 50, cap. & aux.…”

“…All chromatograms were integrated and quantified in XCalibur 3.063 (ThermoFisher Scientific). MS2 spectra were extracted to mzML-format using MSConverter [24] (Peak pick settings: Prefer vendor “check”, MS lvls “1-”; Subset settings: Scan number ““, Scan time ““, Mz win. 0.0-198.10) and compared with the R-script MSMSsim [25] with identities being considered confirmed at a confidence level 1, according to the Schymanski scale [26], when similarity scores > 0.9 and when retention times (RT) in spiked samples matched the samples.…”

“…MS2 spectra were extracted to mzML-format using MSConverter [24] (Peak pick settings: Prefer vendor "check", MS lvls "1-"; Subset settings: Scan number " ", Scan time " ", Mz win. 0.0-198.10) and compared with the R-script MSMSsim [25] with identities being considered confirmed at a (which was not certified by peer review) is the author/funder.…”

Rapid in-plate screening of biotransformation products in single zebrafish embryos

“…Embryo exposures have been described in detail in our prior publication; additional exposures were not carried out for the present work. 36 Briey, single ZF embryos were exposed to six concentrations of PPL (0.050, 0.49, 12, 62, 4550 and 46 540 mg L À1 ; n ¼ 12 embryos per dose), 40 000 mg L À1 of the positive control 3,4-dichloroaniline (12 embryos) and to clean tank water (12 embryos) in a 96-well plate (wp) from 0-120 hours post fertilization (hpf). At termination, exposure water was removed and embryos were subsequently frozen on dry ice, transported to Stockholm University and stored at À80 C. Extraction was carried out by homogenizing the embryos in-plate using mixedsize, stainless steel beads together with a mixture of methanol and chloroform containing a metabolite internal standard (Sphingomyelin ([d18:1/12:0]; see ref.…”

“…At termination, exposure water was removed and embryos were subsequently frozen on dry ice, transported to Stockholm University and stored at À80 C. Extraction was carried out by homogenizing the embryos in-plate using mixedsize, stainless steel beads together with a mixture of methanol and chloroform containing a metabolite internal standard (Sphingomyelin ([d18:1/12:0]; see ref. 36 for details)). Posthomogenization the plates were sonicated and centrifuged prior to placing them directly into the auto-sampler for instrumental analysis.…”