Time-resolved multi-omics reveals staged mitochondrial dysfunction and neurodegeneration-related changes in a tri-culture BTX neurotoxicity model.

Simultaneous benzene, toluene, and xylene (BTX) exposure is a common phenomenon in the workplace and the environment, but has not been well defined by time-resolved molecular events leading to BTX-induced neurotoxicity in multicellular settings. To address these points, we derived an in vitro tri-culture system using SH-SY5Y with a supportive glial compartment (HMC3 + U87) and combined dose-dependent phenotypic profiling with time-resolved transcriptomic, proteomic and metabolic studies after 4, 12, 24, 36 and 48 h of BTX treatment. Working concentrations (IC10, IC20 and IC30) were determined at the end of an initial 24 h dose-response step. Although BTX reduced cell viability in both monoculture and co-culture models, no significant differences in viability were observed between the two models at matched doses. Conversely, the co-culture model had increased sensitivity to sub-lethal toxic responses, which was evidenced by the higher levels of ROS and more obvious concentration-dependent responses to inflammatory, injury and the apoptosis-related markers. Transcriptional pathway dynamics were shown through time-course transcriptomics: initial enrichment of the cell cycle, DNA replication, and p53 signaling; mid-stage metabolic re-programming consisting of HIF-1 signaling, glycolysis/gluconeogenesis and pentose phosphate pathway; and later-stage enrichment of oxidative phosphorylation and Parkin pathways Time-course proteomics and metabolomics respectively indicated a temporal shift into mitochondrial energy dysfunction, proteostasis dysregulation, and neurodegeneration-associated modules. The integrative multi-omics analysis revealed oxidative phosphorylation, Parkinsonism, and thermogenesis as the convergent pathways. Additional evidence of early transcriptional compensation followed by a reduction of mitochondrial and neurofunctional proteins was obtained by time-resolved qPCR and western blot validation. Such results indicate a sequence of BTX neurotoxicity and provide a biologically meaningful multi-omics scheme to study mechanisms underlying and identify biomarkers.