LC-APCI(−)-MS Determination of 1-Chloro-2,4-dinitrobenzene, a Model Substrate for Glutathione S-Transferases

Abstract

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabolic capacity of different test systems. To allow the quantification of CDNB at low, non-toxic concentrations, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negative ion mode. Gas phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/ml in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the common model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (Pac2).

Biotransformation Capacity of Zebrafish (Danio rerio) Early Life Stages: Functionality of the Mercapturic Acid Pathway

“…For measuring the actual exposure concentration used in the toxicity studies, 1 ml of medium was sampled at the beginning of each experiment and stored at -20 °C until further processing. The method for the analysis of CDNB in aqueous solutions and the verification of the exposure concentrations is described in Tierbach et al (2020). The impact of CDNB on zebrafish development was evaluated with the zebrafish embryo toxicity test (zFET) in triplicates, following the OECD guideline 236 (OECD 2013), with some modifications.…”

“…The lethal (increased coagulation, lack of hearth beat, lack of somite formation, non-detachment of the tail,) and sub-lethal (spinal curvature, deformation of the tail, pericardial edema, yolk sac edema) effects observed at 96 hours post fertilization (hpf) were used to calculate the half-maximal lethal concentration (LC 50 ), the half-maximal effective concentration (EC50) and the so called non-toxic concentration, NtC, which is defined as the highest modelled chemical concentration that does not cause toxicity significantly different from the control, with a confidence interval that does not exceed 10% effect and which takes into account the toxicity of each tested replicate (Stadnicka-Michalak et al, 2018). The NtC was chosen as it is a very conservative approach but at the same time it is high enough to be determined by the available quantification methods, which has an LOQ of 17 ng/ml (Tierbach et al, 2020).…”

Feature-Based Molecular Networking Combined with Multivariate Analysis for the Characterization of Glutathione Adducts as a Smoking Gun of Bioactivation

“…Identifying nodes of interest by focusing on the pie chart that presents the ions observed only in the 1 h incubation group seems to be a more effective method for distinguishing GSH-conjugated metabolites than applying the VIP score criterion (Figure B,C). One crucial aspect to consider is the potential for nonenzymatic GSH conjugation, which may occur independently of enzymatic processes and can be overlooked when we solely focus on ions observed only in the incubation group. , To validate the aforementioned cases, the approach was applied to DNCB, a well-known GSH-depleting chemical. , While DNCB itself exhibited poor ionization under HESI conditions, we successfully characterized the GSH adduct of DNCB by identifying the directly connected node ( m / z 474.0925) to GSH node (Figure S3, Supporting Information). Interestingly, the pie chart of the DNCB GSH node included a portion from the 0 h sample, indicating nonenzymatic GSH adduct formation.…”

“…50,51 To validate the aforementioned cases, the approach was applied to DNCB, a well-known GSHdepleting chemical. 52,53 While DNCB itself exhibited poor ionization under HESI conditions, 53 we successfully characterized the GSH adduct of DNCB by identifying the directly connected node (m/z 474.0925) to GSH node (Figure S3, Supporting Information). Interestingly, the pie chart of the DNCB GSH node included a portion from the 0 h sample, indicating nonenzymatic GSH adduct formation.…”

Characterization of the Mercapturic Acid Pathway, an Important Phase II Biotransformation Route, in a Zebrafish Embryo Cell Line

“…To verify CDNB exposure concentrations, CDNB in the exposure medium was analyzed using a quadrupole-orbitrap mass spectrometer (QExactive Plus, Thermo Scientific, United States) and atmospheric pressure chemical ionization in negative ion mode (APCI(−)), based on the method developed by Tierbach et al, using dissociative and nondissociative electron capture ions originating from the CDNB precursor under APCI. Without enrichment, the limit of quantitation was ≥17 ng/mL (83.9 nM).…”