Physiological responses and identification of micro-/trace biochemical markers in bryophytes following exposure to the sulfur mustard mimetic 2-chloroethyl ethyl sulfide

Chemical agents, such as sulfur mustard (SM), are extremely toxic, and prolonged exposure can severely disrupt the metabolism of amino acids and nucleic acids in organisms. To effectively monitor agent exposure and identify specific biomarkers, we employed 2-chloroethyl ethyl sulfide (2-CEES) as a simulant to investigate the changes in metabolic characteristics within three bryophytes under different concentrations of 2-CEES exposure. Key metabolic pathways and enzymes affected by 2-CEES were analysed using theoretical calculations. Results demonstrated significant morphological changes in bryophytes following exposure to 2-CEES. Meanwhile, Chlorophyll fluorescence parameters revealed that 2-CEES markedly disrupted the photosynthetic activity of Physcomitrella patens and Taxiphyllum taxirameum. Metabolomic analysis showed pronounced changes in metabolite expression after 72 hr of 2-CEES (600 mg/m³) treatment across all three species. Pathway enrichment analysis of differentially expressed metabolites (DEMs) indicated that 2-CEES significantly perturbed amino acid, nucleic acid, carbohydrate, and lipid metabolism in Bryum argenteum and Physcomitrella patens. In contrast, Taxiphyllum taxirameum exhibited primary disruptions in lipid metabolism, terpenoid and polyketide metabolism, and membrane transport. Notably, aberrant synthesis of L-Glutamyl-tRNA(Glu) in the aminoacyl-tRNA biosynthesis pathway may correlate with impaired chlorophyll production. In addition, the significant changes of Gamma-Glutamyl-beta-(isoxazolin-5-on-2-yl)alanine, Trans-zeatin riboside, and Cytidine in bryophytes exposed to 600 mg/m³ 2-CEES suggest their potential as micro- and trace biochemical indicators for agent-induced stress. Molecular docking of 2-CEES with key enzymes (Glutathione S-transferase and Glu-tRNA synthetase) revealed that its chloro and methyl groups form hydrogen bonds with residues such as TYR and ARG, interfering with substrate-binding activity and consequently disrupting metabolic pathways.