Chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol elicit MASLD-related lipid metabolic perturbations in HepG2 cells

Metabolic dysfunction-associated steatotic liver disease (MASLD) has been increasingly linked to environmental chemical exposure, yet the metabolic consequences of pesticide parent–metabolite co-exposure remain poorly understood. HepG2 cells were exposed for 48 h to chlorpyrifos (CPF, 10 mg/L), its major metabolite 3,5,6-trichloro-2-pyridinol (TCP, 1.25 mg/L), or their combination (CTCP). Network toxicology was applied for hypothesis generation, followed by integrated lipidomics and transcriptomics analyses. Under the selected exposure conditions, TCP induced greater lipid-droplet accumulation than CPF despite the lower nominal concentration. Lipidomic profiling revealed that both compounds promoted triglyceride accumulation accompanied by extensive remodeling of cholesterol esters, free fatty acids, phospholipids, and sphingolipids. Transcriptomic analysis indicated that CPF elicited a broader response characterized by CYP-mediated xenobiotic metabolism and lipid-handling pathways, whereas TCP exhibited a more restricted signature associated with energy homeostasis and stress-related pathways. Functional assays further supported these distinct responses, showing that TCP exposure reduced ATP levels and increased reactive oxygen species generation, consistent with disrupted cellular energetic and redox homeostasis, while CPF increased cellular CYP1A2 protein levels. Compared with individual exposures, CTCP was associated with greater lipid accumulation and broader transcriptomic and lipidomic alterations involving membrane remodeling, altered cellular energy homeostasis, and changes in inflammation-associated lipid pathways. CPF and TCP produced distinct but overlapping MASLD-relevant lipid metabolic alterations, and their co-exposure produced greater metabolic alterations than either individual exposure under the tested conditions. These findings highlight the importance of considering pesticide metabolites and parent–metabolite co-exposure in environmental health risk assessment.